Reagents and methods useful for detecting diseases of the prostate

ABSTRACT

A set of contiguous and partially overlapping cDNA sequences and polypeptides encoded thereby, designated as PS108 and transcribed from prostate tissue, is described. These sequences are useful for the detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating, or determining the predisposition of an individual to diseases and conditions of the prostate, such as prostate cancer. Also provided are antibodies which specifically bind to PS108-encoded polypeptide or protein, and agonists or inhibitors which prevent action of the tissue-specific PS108 polypeptide, which molecules are useful for the therapeutic treatment of prostate diseases, tumors or metastases.

This APPLN is a DIVISION of U.S. Ser. No. 09/071,710 filed May 1, 1998U.S. Pat. No. 6,130,043 which is a CIP of U.S. Ser. No. 08/850,713 filedMay 2, 1997 ABN.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.08/850,713, filed May 2, 1997, from which priority is claimed pursuantto 35 U.S.C. §120 and which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

This invention relates generally to detecting diseases of the prostate.Furthermore, the invention also relates to reagents and methods fordetecting diseases of the prostate. More particularly, the presentinvention relates to reagents such as polynucleotide sequences and thepolypeptide sequences encoded thereby, as well as methods which utilizethese sequences. The polynucleotide and polypeptide sequences are usefulfor detecting, diagnosing, staging, monitoring, prognosticating, in vivoimaging, preventing or treating, or determining predisposition todiseases or conditions of the prostate such as prostate cancer.

Prostate cancer is the most common form of cancer occurring in males inthe U.S., with projections of 184,500 new cases diagnosed and 39,200related deaths to occur during 1998 (American Cancer Society). Prostatecancer also has shown the largest increase in incidence as compared toother types of cancer, increasing 142% from 1992 to 1996.

Procedures used for detecting, diagnosing, staging, monitoring,prognosticating, in vivo imaging, preventing or treating, or determiningpredisposition to diseases or conditions of the prostate such asprostate cancer are of critical importance to the outcome of thepatient. For example, patients diagnosed with localized prostate cancerhave greater than a 90% five-year survival rate compared to a rate of 25to 31% for patients diagnosed with distant metastasis. (American CancerSociety statistics). A diagnostic procedure for early detection ofprostate cancer should, therefore, specifically detect this disease andbe capable of detecting the presence of prostate cancer before symptomsappear.

Such procedures could include assays based upon the appearance ofvarious disease markers in test samples such as blood, plasma, serum, orurine obtained by minimally invasive procedures which are detectable byimmunological methods. These procedures would provide information to aidthe physician in managing the patient with disease of the prostate andat low cost to the patient. Markers such as the prostate specificantigen (PSA) exist and are used clinically for screening patients forprostate cancer. Elevated levels of PSA protein in serum can be used asa marker in the early detection of prostate cancer in asymptomatic men.G. E. Hanks, et al., In: Cancer: Principles and Practice of Oncology,Vol. 1, Fourth Edition, pp. 1073-1113, Philadelphia, Pa.: J.B.Lippincott Co. (1993.). PSA normally is secreted by the prostate at highlevels into the seminal fluid, but is present in very low levels in theblood of men with normal prostates. However, in patients with diseasesof the prostate including benign prostatic hyperplasia (BPH) andadenocarcinoma of the prostate, the level of PSA can be markedlyelevated in the blood and thus be useful as an indicator of prostatedisease. PSA, however, cannot differentiate between BPH and prostatecancer, which reduces its specificity as a marker for prostate cancer.M. K. Schwartz, et al., In: Cancer: Principlcs and Practice of Oncology,Vol. 1, Fourth Edition, pp. 531-542, Philadelphia, Pa.: J.B. LippincottCo. 1993. New markers which are more specific for prostate cancer thuswould be beneficial in the initial detection of this disease.

A critical step in managing patients with prostate cancer is thepresurgical staging of the cancer to provide prognostic value andcriteria for designing optimal therapy. Improved procedures foraccurately staging prostate cancer prior to surgery are needed. Onestudy demonstrated that current methods of staging prostate cancer priorto surgery were incorrect approximately fifty percent (50%) of the time.F. Labrie, et al., Urology 44 (Symposium Issue): 29-37 (1994). Prostatecancer management also could be improved by utilizing new markers foundin an inappropriate body compartment. Such markers could be mRNA orprotein markers expressed by cells originating from the primary prostatetumor but residing in blood, bone marrow or lymph nodes and could besensitive indicators for metastasis to these distal organs. For example,in patients with metastatic prostate cancer, PSA protein has beendetected by immunohistochemical techniques in bone marrow, and PSA mRNAhas been detected by RT-PCR in cells of blood, lymph nodes and bonemarrow. K. Pantel, et al., Onkologie 18: 394-401 (1995).

New markers which could predict the biologic behavior of early prostatecancers would also be of significant value. Early prostate cancers thatthreaten or will threaten the life of the patient are more clinicallyimportant than those that do not or will not be a threat. G. E. Hanks,supra. A need therefore exists for new markers which can differentiatebetween the clinically important and unimportant prostate cancers. Suchmarkers would allow the clinician to accurately identify and effectivelytreat early cancers localized to the prostate which could otherwisemetastasize and kill the patient. Further, if one could show that such amarker characteristic of aggressive cancer was absent, the patient couldbe spared expensive and non-beneficial treatment.

It also would be beneficial to find a prostate associated marker whichis more sensitive in detecting recurrence of prostate cancer than PSAand which is not affected by androgens. To date, PSA has proven to bethe most sensitive marker for detecting recurrent disease. However, insome cases tumor progression occurs without PSA elevation due tohormonal therapy utilized for treating the cancer. Although the decreasein androgen results in a concomitant decrease in PSA, it does notnecessarily reflect a decrease in tumor metastasis. This complication isthe result of androgen-stimulated PSA expression. Part of the decline inPSA observed after androgen ablation is due not to tumor cell death butto diminished PSA expression. G. E. Hanks, supra.

It therefore would be advantageous to provide specific methods andreagents for detecting, diagnosing, staging, monitoring,prognosticating, in vivo imaging, preventing or treating, or determiningpredisposition to diseases and conditions of the prostate, or toindicate possible predisposition to these conditions. Such methods wouldinclude assaying a test sample for products of a gene which areoverexpressed in prostate diseases and conditions such as cancer. Suchmethods may also include assaying a test sample for products of a genealteration associated with prostate disease or condition. Such methodsmay further include assaying a test sample for products of a gene whosedistribution among the various tissues and compartments of the body havebeen altered by a prostate-associated disease or condition such ascancer. Useful reagents include polynucleotide(s), or fragment(s)thereof which may be used in diagnostic methods such as reversetranscriptase-polymerase chain reaction (RT-PCR), PCR, or hybridizationassays of mRNA extracted from biopsied tissue, blood or other testsamples; polypeptides or proteins which are the translation products ofsuch mRNAs; or antibodies directed against these polypeptides orproteins. Drug treatment or gene therapy for diseases or conditions ofthe prostate can then be based on these identified gene sequences ortheir expressed proteins and efficacy of any particular therapy can bemonitored. Furthermore, it would be advantageous to have availablealternative, non-surgical diagnostic methods capable of detecting earlystage prostate disease such as cancer.

SUMMARY OF THE INVENTION

The present invention provides a method of detecting a target PS108polynucleotide in a test sample, which method comprises contacting thetest sample with at least one PS108-specific polynucleotide anddetecting the presence of the target PS108 polynucleotide in the testsample. The PS108-specific polynucleotide has at least 50% identity witha polynucleotide selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, SEQUENCE ID NO 5,SEQUENCE ID NO 6, SEQUENCE ID NO 7, SEQUENCE ID NO 8, SEQUENCE ID NO 9,SEQUENCE ID NO 10, SEQUENCE ID NO 11, SEQUENCE ID NO 12, SEQUENCE ID NO13, SEQUENCE ID NO 14, SEQUENCE ID NO 15, SEQUENCE ID NO 16 (“SEQUENCEID NOS 1-16”), and fragments or complements thereof. Also, thePS108-specific polynucleotide may be attached to a solid phase prior toperforming the method.

The present invention also provides a method for detecting PS108 mRNA ina test sample, which comprises performing reverse transcription (RT)with at least one primer in order to produce cDNA, amplifying the cDNAso obtained using PS108 oligonucleotides as sense and antisense primersto obtain PS108 amplicon, and detecting the presence of the PS108amplicon as an indication of the presence of PS108 mRNA in the testsample, wherein the PS108 oligonucleotides have at least 50% identity toa sequence selected from the group consisting of SEQUENCE ID NOS 1-16,and fragments or complements thereof. Amplification can be performed bythe polymerase chain reaction. Also, the test sample can be reacted witha solid phase prior to performing the method, prior to amplification orprior to detection. This reaction can be a direct or an indirectreaction. Further, the detection step can comprise utilizing adetectable label capable of generating a measurable signal. Thedetectable label can be attached to a solid phase.

The present invention further provides a method of detecting a targetPS108 polynucleotide in a test sample suspected of containing targetPS108 polynucleotides, which comprises (a) contacting the test samplewith at least one PS108 oligonucleotide as a sense primer and at leastone PS108 oligonucleotide as an anti-sense primer, and amplifying sameto obtain a first stage reaction product; (b) contacting the first stagereaction product with at least one other PS108 oligonucleotide to obtaina second stage reaction product, with the proviso that the other PS108oligonucleotide is located 3′ to the PS108 oligonucleotides utilized instep (a) and is complementary to the first stage reaction product; and(c) detecting the second stage reaction product as an indication of thepresence of a target PS108 polynucleotide in the test sample. The PS108oligonucleotides selected as reagents in the method have at least 50%identity with a sequence selected from the group consisting of SEQUENCEID NOS 1-16, and fragments or complements thereof. Amplification may beperformed by the polymerase chain reaction. The test sample can bereacted either directly or indirectly with a solid phase prior toperforming the method, or prior to amplification, or prior to detection.The detection step also comprises utilizing a detectable label capableof generating a measurable signal; further, the detectable label can beattached to a solid phase. Test kits useful for detecting target PS108polynucleotides in a test sample are also provided which comprise acontainer containing at least one PS108-specific polynucleotide selectedfrom the group consisting of SEQUENCE ID NOS 1-16, and fragments orcomplements thereof. These test kits further comprise containers withtools useful for collecting test samples (such as, for example, blood,urine, saliva and stool). Such tools include lancets and absorbent paperor cloth for collecting and stabilizing blood; swabs for collecting andstabilizing saliva; and cups for collecting and stabilizing urine orstool samples. Collection materials, such as papers, cloths, swabs,cups, and the like, may optionally be treated to avoid denaturation orirreversible adsorption of the sample. The collection materials also maybe treated with or contain preservatives, stabilizers or antimicrobialagents to help maintain the integrity of the specimens.

The present invention also provides a purified polynucleotide orfragment thereof derived from a PS108 gene. The purified polynucleotideis capable of selectively hybridizing to the nucleic acid of the PS108gene, or a complement thereof. The polynucleotide has at least 50%identity with a polynucleotide selected from the group consisting ofSEQUENCE ID NOS 1-16, and fragments or complements thereof. Further, thepurified polynucleotide can be produced by recombinant and/or synthetictechniques. The purified recombinant polynucleotide can be containedwithin a recombinant vector. The invention further comprises a host celltransfected with the recombinant vector.

The present invention further provides a recombinant expression systemcomprising a nucleic acid sequence that includes an open reading framederived from PS108. The nucleic acid sequence has at least 50% identitywith a sequence selected from the group consisting of SEQUENCE ID NOS1-16, and fragments or complements thereof. The nucleic acid sequence isoperably linked to a control sequence compatible with a desired host.Also provided is a cell transfected with this recombinant expressionsystem.

The present invention also provides a polypeptide encoded by PS108. Thepolypeptide can be produced by recombinant technology, provided inpurified form, or produced by synthetic techniques. The polypeptidecomprises an amino acid sequence which has at least 50% identity with anamino acid sequence selected from At the group consisting of SEQUENCE IDNO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38, SEQUENCE ID NO 39, andfragments thereof.

Also provided is an antibody which specifically binds to at least onePS108 epitope. The antibody can be a polyclonal or monoclonal antibody.The epitope is derived from an amino acid sequence selected from thegroup consisting of SEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO38, SEQUENCE ID NO 39, and fragments thereof. Assay kits for determiningthe presence of PS108 antigen or anti-PS108 antibody in a test sampleare also included. In one embodiment, the assay kits comprise acontainer containing at least one PS108 polypeptide having at least 50%identity with an amino acid sequence selected from the group consistingof SEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38, SEQUENCE IDNO 39, and fragments thereof. Further, the test kit can comprise acontainer with tools useful for collecting test samples (such as blood,urine, saliva, and stool). Such tools include lancets and absorbentpaper or cloth for collecting and stabilizing blood; swabs forcollecting and stabilizing saliva; and cups for collecting andstabilizing urine or stool samples. Collection materials such as papers,cloths, swabs, cups, and the like, may optionally be treated to avoiddenaturation or irreversible adsorption of the sample. These collectionmaterials also may be treated with or contain preservatives, stabilizersor antimicrobial agents to help maintain the integrity of the specimens.Also, the polypeptide can be attached to a solid phase.

In another embodiment of the invention, antibodies or fragments thereofagainst the PS108 antigen can be used to detect or image localization ofthe antigen in a patient for the purpose of detecting or diagnosing adisease or condition. Such antibodies can be polyclonal or monoclonal,or made by molecular biology techniques, and can be labeled with avariety of detectable labels, including but not limited to radioisotopesand paramagnetic metals. Furthermore, antibodies or fragments thereof,whether monoclonal, polyclonal, or made by molecular biology techniques,can be used as therapeutic agents for the treatment of diseasescharacterized by expression of the PS108 antigen. In the case oftherapeutic applications, the antibody may be used withoutderivitization, or it may be derivitized with a cytotoxic agent such asa radioisotope, enzyme, toxin, drug, prodrug, or the like.

Another assay kit for determining the presence of PS108 antigen oranti-PS108 antibody in a test sample comprises a container containing anantibody which specifically binds to a PS108 antigen, wherein the PS108antigen comprises at least one PS108-encoded epitope. The PS108 antigenhas at least about 60% sequence similarity to a sequence of aPS108-encoded antigen selected from the group consisting of SEQUENCE IDNO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38, SEQUENCE ID NO 39, andfragments thereof. These test kits can further comprise containers withtools useful for collecting test samples (such as blood, urine, saliva,and stool). Such tools include lancets and absorbent paper or cloth forcollecting and stabilizing blood; swabs for collecting and stabilizingsaliva; cups for collecting and stabilizing urine or stool samples.Collection materials, papers, cloths, swabs, cups and the like, mayoptionally be treated to avoid denaturation or irreversible adsorptionof the sample. These collection materials also may be treated with, orcontain, preservatives, stabilizers or antimicrobial agents to helpmaintain the integrity of the specimens. The antibody can be attached toa solid phase.

A method for producing a polypeptide which contains at least one epitopeof PS108 is provided, which method comprises incubating host cellstransfected with an expression vector. This vector comprises apolynucleotide sequence encoding a polypeptide, wherein the polypeptidecomprises an amino acid sequence having at least 50% identity with aPS108 amino acid sequence selected from the group consisting of SEQUENCEID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38, SEQUENCE ID NO 39, andfragments thereof.

A method for detecting PS108 antigen in a test sample suspected ofcontaining PS108 antigen also is provided. The method comprisescontacting the test sample with an antibody or fragment thereof whichspecifically binds to at least one epitope of PS108 antigen, for a timeand under conditions sufficient for the formation of antibody/antigencomplexes; and detecting the presence of such complexes containing theantibody as an indication of the presence of PS108 antigen in the testsample. The antibody can be attached to a solid phase and may be eithera monoclonal or polyclonal antibody. Furthermore, the antibodyspecifically binds to at least one PS108 antigen selected from the groupconsisting of SEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38,SEQUENCE ID NO 39, and fragments thereof.

Another method is provided which detects antibodies which specificallybind to PS108 antigen in a test sample suspected of containing theseantibodies. The method comprises contacting the test sample with apolypeptide which contains at least one PS108 epitope, wherein the PS108epitope comprises an amino acid sequence having at least 50% identitywith an amino acid sequence encoded by a PS108 polynucleotide, or afragment thereof. Contacting is carried out for a time and underconditions sufficient to allow antigen/antibody complexes to form. Themethod further entails detecting complexes which contain thepolypeptide. The polypeptide can be attached to a solid phase. Further,the polypeptide can be a recombinant protein or a synthetic peptidehaving at least 50% identity with an amino acid sequence selected fromthe group consisting of SEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCEID NO 38, SEQUENCE ID NO 39, and fragments thereof.

The present invention provides a cell transfected with a PS108 nucleicacid sequence that encodes at least one epitope of a PS108 antigen, orfragment thereof. The nucleic acid sequence is selected from the groupconsisting of SEQUENCE ID NOS 1-16, and fragments or complementsthereof.

A method for producing antibodies to PS108 antigen also is provided,which method comprises administering to an individual an isolatedimmunogenic polypeptide or fragment thereof, wherein the isolatedimmunogenic polypeptide comprises at least one PS108 epitope. Theimmunogenic polypeptide is administered in an amount sufficient toproduce an immune response. The isolated, immunogenic polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38, SEQUENCE ID NO39, and fragments thereof.

Another method for producing antibodies which specifically bind to PS108antigen is disclosed, which method comprises administering to anindividual a plasmid comprising a nucleic acid sequence which encodes atleast one PS108 epitope derived from an amino acid sequence selectedfrom the group consisting of SEQUENCE ID NO 36, SEQUENCE ID NO 37,SEQUENCE ID NO 38, SEQUENCE ID NO 39, and fragments thereof. The plasmidis administered in an amount such that the plasmid is taken up by cellsin the individual and expressed at levels sufficient to produce animmune response.

Also provided is a composition of matter that comprises a PS108polynucleotide of at least about 10-12 nucleotides having at least 50%identity with a polynucleotide selected from the group consisting ofSEQUENCE ID NOS 1-16, and fragments or complements thereof. The PS108polynucleotide encodes an amino acid sequence having at least one PS108epitope. Another composition of matter provided by the present inventioncomprises a polypeptide with at least one PS108 epitope of about 8-10amino acids. The polypeptide comprises an amino acid sequence having atleast 50% identity with an amino acid sequence selected from the groupconsisting of SEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38,SEQUENCE ID NO 39, and fragments thereof. Also provided is a gene, orfragment thereof, coding for a PS108 polypeptide which has at least 50%identity to SEQUENCE ID NO 36; and a gene, or a fragment thereof,comprising DNA having at least 50% identity with SEQUENCE ID NO 15 orSEQUENCE ID NO 16.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show the nucleotide alignment of clones 1864683 (SEQUENCE IDNO 1), 1711346 (SEQUENCE ID NO 2), 1913982 (SEQUENCE ID NO 3), 825779(SEQUENCE ID NO 4), 2626650 (SEQUENCE ID NO 5), 1808389 (SEQUENCE ID NO6), 1651121 (SEQUENCE ID NO 7), 3520833 (SEQUENCE ID NO 8), 2188949(SEQUENCE ID NO 9), 3497504 (SEQUENCE ID NO 10), 3964174 (SEQUENCE ID NO11), 3705332 (SEQUENCE ID NO 12), 2270646 (SEQUENCE ID NO 13), 1810610(SEQUENCE ID NO 14), the full-length sequence of clone 1711346(designated as clone 1711346IH (SEQUENCE ID NO 15)), and the consensussequence (SEQUENCE ID NO 16) derived therefrom.

FIG. 2 shows the contig map depicting the formation of the consensusnucleotide sequence (SEQUENCE ID NO 16) from the nucleotide alignment ofoverlapping clones 1864683 (SEQUENCE ID NO 1), 1711346 (SEQUENCE ID NO2), 1913982 (SEQUENCE ID NO 3), 825779 (SEQUENCE ID NO 4), 2626650(SEQUENCE ID NO 5), 1808389 (SEQUENCE ID NO 6), 1651121 (SEQUENCE ID NO7), 3520833 (SEQUENCE ID NO 8), 2188949 (SEQUENCE ID NO 9),3497504(SEQUENCE ID NO 10), 3964174 (SEQUENCE ID NO 11), 3705332 (SEQUENCE IDNO 12), 2270646 (SEQUENCE ID NO 13), 1810610 (SEQUENCE ID NO 14), and1711346IH (SEQUENCE ID NO 15).

FIG. 3A is a scan of a SYBR® Green-stained agarose gel of PS108RNA-specific RT-PCR amplification products from prostate tissue RNAs.FIG. 3B is a scan of a SYBR® Green-stained agarose gel of PS108RNA-specific RT-PCR amplification products of RNAs from normal orcancerous placenta, colon, breast, prostate, and lung tissues.

FIG. 4 shows the results of a Western blot performed on a panel oftissue protein extracts detected with antiserum against the PS108synthetic peptide of SEQUENCE ID NO 37.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a gene, or a fragment thereof, whichcodes for a PS108 polypeptide having at least about 50% identity toSEQUENCE ID NO 36. The present invention further encompasses a PS108gene, or a fragment thereof, comprising DNA which has at least about 50%identity with SEQUENCE ID NO 15 or SEQUENCE ID NO 16.

The present invention also provides methods for assaying a test samplefor products of a prostate tissue gene designated as PS108, whichcomprises making cDNA from mRNA in the test sample, and detecting thecDNA as an indication of the presence of prostate tissue gene PS108. Themethod may include an amplification step, wherein one or more portionsof the mRNA from PS108 corresponding to the gene or fragments thereof,is amplified. Methods also are provided for assaying for the translationproducts of PS108. Test samples which may be assayed by the methodsprovided herein include tissues, cells, body fluids and secretions. Thepresent invention also provides reagents such as oligonucleotide primersand polypeptides which are useful in performing these methods.

Portions of the nucleic acid sequences disclosed herein are useful asprimers for the reverse transcription of RNA or for the amplification ofcDNA; or as probes to determine the presence of certain mRNA sequencesin test samples. Also disclosed are nucleic acid sequences which permitthe production of encoded polypeptide sequences which are useful asstandards or reagents in diagnostic immunoassays, as targets forpharmaceutical screening assays and/or as components or as target sitesfor various therapies. Monoclonal and polyclonal antibodies directedagainst at least one epitope contained within these polypeptidesequences are useful as delivery agents for therapeutic agents as wellas for diagnostic tests and for screening for diseases or conditionsassociated with PS108, especially prostate cancer. Isolation ofsequences of other portions of the gene of interest can be accomplishedutilizing probes or PCR primers derived from these nucleic acidsequences. This allows additional probes of the mRNA or cDNA of interestto be established, as well as corresponding encoded polypeptidesequences. These additional molecules are useful in detecting,diagnosing, staging, monitoring, prognosticating, in vivo imaging,preventing or treating, or determining the predisposition to diseasesand conditions of the prostate, such as prostate cancer, characterizedby PS108, as disclosed herein.

Techniques for determining amino acid sequence “similarity” arewell-known in the art. In general, “similarity” means the exact aminoacid to amino acid comparison of two or more polypeptides at theappropriate place, where amino acids are identical or possess similarchemical and/or physical properties such as charge or hydrophobicity. Aso-termed “percent similarity” then can be determined between thecompared polypeptide sequences. Techniques for determining nucleic acidand amino acid sequence identity also are well known in the art andinclude determining the nucleotide sequence of the mRNA for that gene(usually via a cDNA intermediate) and determining the amino acidsequence encoded thereby, and comparing this to a second amino acidsequence. In general, “identity” refers to an exact nucleotide tonucleotide or amino acid to amino acid correspondence of twopolynucleotides or polypeptide sequences, respectively. Two or morepolynucleotide sequences can be compared by determining their “percentidentity.” Two or more amino acid sequences likewise can be compared bydetermining their “percent identity.” The programs available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.), for example, the GAP program, arecapable of calculating both the identity between two polynucleotides andthe identity and similarity between two polypeptide sequences,respectively. Other programs for calculating identity or similaritybetween sequences are known in the art.

The compositions and methods described herein will enable theidentification of certain markers as indicative of a prostate tissuedisease or condition; the information obtained therefrom will aid in thedetecting, diagnosing, staging, monitoring, prognosticating, in vivoimaging, preventing or treating, or determining diseases or conditionsassociated with PS108, especially prostate cancer. Test methods include,for example, probe assays which utilize the sequencers) provided hereinand which also may utilize nucleic acid amplification methods such asthe polymerase chain reaction (PCR), the ligase chain reaction (LCR),and hybridization. In addition, the nucleotide sequences provided hereincontain open reading frames from which an immunogenic epitope may befound. This epitope is believed to be unique to the disease state orcondition associated with PS108. It also is thought that thepolynucleotides or polypeptides and protein encoded by the PS108 geneare useful as a marker. This marker is either elevated in disease suchas prostate cancer, altered in disease such as prostate cancer, orpresent as a normal protein but appearing in an inappropriate bodycompartment. The uniqueness of the epitope may be determined by (i) itsimmunological reactivity and specificity with antibodies directedagainst proteins and polypeptides encoded by the PS108 gene, and (ii)its nonreactivity with any other tissue markers. Methods for determiningimmunological reactivity are well-known and include, but are not limitedto, for example, radioimmunoassay (RIA), enzyme-linked immunoabsorbentassay (ELISA), hemagglutination (HA), fluorescence polarizationimmunoassay (FPIA), chemiluminescent immunoassay (CLIA) and others.Several examples of suitable methods are described herein.

Unless otherwise stated, the following terms shall have the followingmeanings:

A polynucleotide “derived from” or “specific for” a designated sequencerefers to a polynucleotide sequence which comprises a contiguoussequence of approximately at least about 6 nucleotides, preferably atleast about 8 nucleotides, more preferably at least about 10-12nucleotides, and even more preferably at least about 15-20 nucleotidescorresponding, i.e., identical or complementary to, a region of thedesignated nucleotide sequence. The sequence may be complementary oridentical to a sequence which is unique to a particular polynucleotidesequence as determined by techniques known in the art. Comparisons tosequences in databanks, for example, can be used as a method todetermine the uniqueness of a designated sequence. Regions from whichsequences may be derived, include but are not limited to, regionsencoding specific epitopes, as well as non-translated and/ornon-transcribed regions.

The derived polynucleotide will not necessarily be derived physicallyfrom the nucleotide sequence of interest under study, but may begenerated in any manner, including, but not limited to, chemicalsynthesis, replication, reverse transcription or transcription, which isbased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived. As such, it mayrepresent either a sense or an antisense orientation of the originalpolynucleotide. In addition, combinations of regions corresponding tothat of the designated sequence may be modified in ways known in the artto be consistent with the intended use.

A “fragment” of a specified polynucleotide refers to a polynucleotidesequence which comprises a contiguous sequence of approximately at leastabout 6 nucleotides, preferably at least about 8 nucleotides, morepreferably at least about 10-12 nucleotides, and even more preferably atleast about 15-20 nucleotides corresponding, i.e., identical orcomplementary to, a region of the specified nucleotide sequence.

The term “primer” denotes a specific oligonucleotide sequence which iscomplementary to a target nucleotide sequence and used to hybridize tothe target nucleotide sequence. A primer serves as an initiation pointfor nucleotide polymerization catalyzed by either DNA polymerase, RNApolymerase or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment (or nucleotideanalog segment, e.g., PNA as defined hereinbelow) which can be used toidentify a specific polynucleotide present in samples bearing thecomplementary sequence.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences which areimmunologically identifiable with a polypeptide encoded by the sequence.Thus, a “polypeptide,” “protein,” or “amino acid” sequence has at leastabout 50% identity, preferably about 60% identity, more preferably about75-85% identity, and most preferably about 90-95% or mote identity to aPS108 amino acid sequence. Further, the PS108 “polypeptide,” “protein,”or “amino acid” sequence may have at least about 60% similarity,preferably at least about 75% similarity, more preferably about 85%similarity, and most preferably about 95% or more similarity to apolypeptide or amino acid sequence of PS108. This amino acid sequencecan be selected from the group consisting of SEQUENCE ID NO 36, SEQUENCEID NO 37, SEQUENCE ID NO 38, SEQUENCE ID NO 39, and fragments thereof.

A “recombinant polypeptide,” “recombinant protein,” or “a polypeptideproduced by recombinant techniques,” which terms may be usedinterchangeably herein, describes a polypeptide which by virtue of itsorigin or manipulation is not associated with all or a portion of thepolypeptide with which it is associated in nature and/or is linked to apolypeptide other than that to which it is linked in nature. Arecombinant or encoded polypeptide or protein is not necessarilytranslated from a designated nucleic acid sequence. It also may begenerated in any manner, including chemical synthesis or expression of arecombinant expression system.

The term “synthetic peptide” as used herein means a polymeric form ofamino acids of any length, which may be chemically synthesized bymethods well-known to the routineer. These synthetic peptides are usefulin various applications.

The term “polynucleotide” as used herein means a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes double- and single-stranded DNA,as well as double- and single-stranded RNA. It also includesmodifications, such as methylation or capping and unmodified forms ofthe polynucleotide. The terms “polynucleotide,” “oligomer,”“oligonucleotide,” and “oligo” are used interchangeably herein.

“A sequence corresponding to a cDNA” means that the sequence contains apolynucleotide sequence that is identical or complementary to a sequencein the designated DNA. The degree (or “percent”) of identity orcomplementarity to the cDNA will be approximately 50% or greater,preferably at least about 70% or greater, and more preferably at leastabout 90% or greater. The sequence that corresponds to the identifiedcDNA will be at least about 50 nucleotides in length, preferably atleast about 60 nucleotides in length, and more preferably at least about70 nucleotides in length. The correspondence between the gene or genefragment of interest and the cDNA can be determined by methods known inthe art and include, for example, a direct comparison of the sequencedmaterial with the cDNAs described, or hybridization and digestion withsingle strand nucleases, followed by size determination of the digestedfragments.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

“Purified polypeptide” or “purified protein” means a polypeptide ofinterest or fragment thereof which is essentially free of, e.g.,contains less than about 50%, preferably less than about 70%, and morepreferably less than about 90%, cellular components with which thepolypeptide of interest is naturally associated. Methods for purifyingpolypeptides of interest are known in the art.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, which is separated from some orall of the coexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

“Polypeptide” and “protein” are used interchangeably herein and indicateat least one molecular chain of amino acids linked through covalentand/or non-covalent bonds. The terms do not refer to a specific lengthof the product. Thus peptides, oligopeptides and proteins are includedwithin the definition of polypeptide. The terms includepost-translational modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Inaddition, protein fragments, analogs, mutated or variant proteins,fusion proteins and the like are included within the meaning ofpolypeptide.

A “fragment” of a specified polypeptide refers to an amino acid sequencewhich comprises at least about 3-5 amino acids, more preferably at leastabout 8-10 amino acids, and even more preferably at least about 15-20amino acids derived from the specified polypeptide.

“Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cellcultures,” and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be, or have been, used as recipients for recombinant vector orother transferred DNA, and include the original progeny of the originalcell which has been transfected.

As used herein “replicon” means any genetic element, such as a plasmid,a chromosome or a virus, that behaves as an autonomous unit ofpolynucleotide replication within a cell.

A “vector” is a replicon in which another polynucleotide segment isattached, such as to bring about the replication and/or expression ofthe attached segment.

The term “control sequence” refers to a polynucleotide sequence which isnecessary to effect the expression of a coding sequence to which it isligated. The nature of such control sequences differs depending upon thehost organism. In prokaryotes, such control sequences generally includea promoter, a ribosomal binding site and terminators; in eukaryotes,such control sequences generally include promoters, terminators and, insome instances, enhancers. The term “control sequence” thus is intendedto include at a minimum all components whose presence is necessary forexpression, and also may include additional components whose presence isadvantageous, for example, leader sequences.

“Operably linked” refers to a situation wherein the components describedare in a relationship permitting them to function in their intendedmanner. Thus, for example, a control sequence “operably linked” to acoding sequence is ligated in such a manner that expression of thecoding sequence is achieved under conditions compatible with the controlsequence.

The term “open reading frame” or “ORF” refers to a region of apolynucleotide sequence which encodes a polypeptide. This region mayrepresent a portion of a coding sequence or a total coding sequence.

A “coding sequence” is a polynucleotide sequence which is transcribedinto mRNA and translated into a polypeptide when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. A codingsequence can include, but is not limited to, mRNA, cDNA and recombinantpolynucleotide sequences.

The term “immunologically identifiable with/as” refers to the presenceof epitope(s) and polypeptide(s) which also are present in and areunique to the designated polypeptide(s). Immunological identity may bedetermined by antibody binding and/or competition in binding. Thesetechniques are known to the routineer and also are described herein. Theuniqueness of an epitope also can be determined by computer searches ofknown data banks, such as GenBank, for the polynucleotide sequence whichencodes the epitope and by amino acid sequence comparisons with otherknown proteins.

As used herein, “epitope” means an antigenic determinant of apolypeptide or protein. Conceivably, an epitope can comprise three aminoacids in a spatial conformation which is unique to the epitope.Generally, an epitope consists of at least five such amino acids andmore usually, it consists of at least eight to ten amino acids. Methodsof examining spatial conformation are known in the art and include, forexample, x-ray crystallography and two-dimensional nuclear magneticresonance.

A “conformational epitope” is an epitope that is comprised of a specificjuxtaposition of amino acids in an immunologically recognizablestructure, such amino acids being present on the same polypeptide in acontiguous or non-contiguous order or present on different polypeptides.

A polypeptide is “immunologically reactive” with an antibody when itbinds to an antibody due to antibody recognition of a specific epitopecontained within the polypeptide. Immunological reactivity may bedetermined by antibody binding, more particularly, by the kinetics ofantibody binding, and/or by competition in binding using ascompetitor(s) a known polypeptide(s) containing an epitope against whichthe antibody is directed. The methods for determining whether apolypeptide is immunologically reactive with an antibody are known inthe art.

As used herein, the term “immunogenic polypeptide containing an epitopeof interest” means naturally occurring polypeptides of interest orfragments thereof, as well as polypeptides prepared by other means, forexample, by chemical synthesis or the expression of the polypeptide in arecombinant organism.

The term “transfection” refers to the introduction of an exogenouspolynucleotide into a prokaryotic or eucaryotic host cell, irrespectiveof the method used for the introduction. The term “transfection” refersto both stable and transient introduction of the polynucleotide, andencompasses direct uptake of polynucleotides, transformation,transduction, and f-mating. Once introduced into the host cell, theexogenous polynucleotide may be maintained as a non-integrated replicon,for example, a plasmid, or alternatively, may be integrated into thehost genome.

“Treatment” refers to prophylaxis and/or therapy.

The term “individual” as used herein refers to vertebrates, particularlymembers of the mammalian species and includes, but is not limited to,domestic animals, sports animals, primates and humans; moreparticularly, the term refers to humans.

The term “sense strand” or “plus strand” (or “+”) as used herein denotesa nucleic acid that contains the sequence that encodes the polypeptide.The term “antisense strand” or “minus strand” (or “−”) denotes a nucleicacid that contains a sequence that is complementary to that of the“plus” strand.

The term “test sample” refers to a component of an individual's bodywhich is the source of the analyte (such as antibodies of interest orantigens of interest). These components are well known in the art. Atest sample is typically anything suspected of containing a targetsequence. Test samples can be prepared using methodologies well known inthe art such as by obtaining a specimen from an individual and, ifnecessary, disrupting any cells contained thereby to release targetnucleic acids. These test samples include biological samples which canbe tested by the methods of the present invention described herein andinclude human and animal body fluids such as whole blood, serum, plasma,cerebrospinal fluid, sputum, bronchial washing, bronchial aspirates,urine, lymph fluids, and various external secretions of the respiratory,intestinal and genitourinary tracts, tears, saliva, milk, white bloodcells, myelomas and the like; biological fluids such as cell culturesupernatants; tissue specimens which may be fixed; and cell specimenswhich may be fixed.

“Purified product” refers to a preparation of the product which has beenisolated from the cellular constituents with which the product isnormally associated and from other types of cells which may be presentin the sample of interest.

“PNA” denotes a “peptide nucleic acid analog” which may be utilized in aprocedure such as an assay described herein to determine the presence ofa target. “MA” denotes a “morpholino analog” which may be utilized in aprocedure such as an assay described herein to determine the presence ofa target. See, for example, U.S. Pat. No. 5,378,841, which isincorporated herein by reference. PNAs are neutrally charged moietieswhich can be directed against RNA targets or DNA. PNA probes used inassays in place of, for example, the DNA probes of the presentinvention, offer advantages not achievable when DNA probes are used.These advantages include manufacturability, large scale labeling,reproducibility, stability, insensitivity to changes in ionic strengthand resistance to enzymatic degradation which is present in methodsutilizing DNA or RNA. These PNAs can be labeled with (“attached to”)such signal generating compounds as fluorescein, radionucleotides,chemiluminescent compounds and the like. PNAs or other nucleic acidanalogs such as MAs thus can be used in assay methods in place of DNA orRNA. Although assays are described herein utilizing DNA probes, it iswithin the scope of the routineer that PNAs or MAs can be substitutedfor RNA or DNA with appropriate changes if and as needed in assayreagents.

“Analyte,” as used herein, is the substance to be detected which may bepresent in the test sample. The analyte can be any substance for whichthere exists a naturally occurring specific binding member (such as anantibody), or for which a specific binding member can be prepared. Thus,an analyte is a substance that can bind to one or more specific bindingmembers in an assay. “Analyte” also includes any antigenic substances,haptens, antibodies and combinations thereof. As a member of a specificbinding pair, the analyte can be detected by means of naturallyoccurring specific binding partners (pairs) such as the use of intrinsicfactor protein as a member of a specific binding pair for thedetermination of Vitamin B12, the use of folate-binding protein todetermine folic acid, or the use of a lectin as a member of a specificbinding pair for the determination of a carbohydrate. The analyte caninclude a protein, a polypeptide, an amino acid, a nucleotide target andthe like. The analyte can be soluble in a body fluid such as blood,blood plasma or serum, urine or the like. The analyte can be in atissue, either on a cell surface or within a cell. The analyte can be onor in a cell dispersed in a body fluid such as blood, urine, breastaspirate, or obtained as a biopsy sample.

The terms “diseases of the prostate,” “prostate disease,” and “conditionof the prostate” are used interchangeably herein to refer to any diseaseor condition of the prostate including, but not limited to, benignprostatic hyperplasia (BPH), prostatitis, prostatic intraepithelialneoplasia (PIN) and cancer.

“Prostate cancer,” as used herein, refers to any malignant disease ofthe prostate including, but not limited to, adenocarcinoma, small cellundifferentiated carcinoma and mucinous (colloid) cancer.

An “Expressed Sequence Tag” or “EST” refers to the partial sequence of acDNA insert which has been made by reverse transcription of mRNAextracted from a tissue followed by insertion into a vector.

A “transcript image” refers to a table or list giving the quantitativedistribution of ESTs in a library and represents the genes active in thetissue from which the library was made.

The present invention provides assays which utilize specific bindingmembers. A “specific binding member,” as used herein, is a member of aspecific binding pair. That is, two different molecules where one of themolecules, through chemical or physical means, specifically binds to thesecond molecule. Therefore, in addition to antigen and antibody specificbinding pairs of common immunoassays, other specific binding pairs caninclude biotin and avidin, carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzyme inhibitors, and enzymes and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, antibodies and antibody fragments, both monoclonal andpolyclonal and complexes thereof, including those formed by recombinantDNA molecules.

The term “hapten,” as used herein, refers to a partial antigen ornon-protein binding member which is capable of binding to an antibody,but which is not capable of eliciting antibody formation unless coupledto a carrier protein.

A “capture reagent,” as used herein, refers to an unlabeled specificbinding member which is specific either for the analyte as in a sandwichassay, for the indicator reagent or analyte as in a competitive assay,or for an ancillary specific binding member, which itself is specificfor the analyte, as in an indirect assay. The capture reagent can bedirectly or indirectly bound to a solid phase material before theperformance of the assay or during the performance of the assay, therebyenabling the separation of immobilized complexes from the test sample.

The “indicator reagent” comprises a “signal-generating compound”(“label”) which is capable of generating and generates a measurablesignal detectable by external means, conjugated (“attached”) to aspecific binding member. In addition to being an antibody member of aspecific binding pair, the indicator reagent also can be a member of anyspecific binding pair, including either hapten-anti-hapten systems suchas biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin,a complementary nucleotide sequence, an effector or a receptor molecule,an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme andthe like. An immunoreactive specific binding member can be an antibody,an antigen, or an antibody/antigen complex that is capable of bindingeither to the polypeptide of interest as in a sandwich assay, to thecapture reagent as in a competitive assay, or to the ancillary specificbinding member as in an indirect assay. When describing probes and probeassays, the term “reporter molecule” may be used. A reporter moleculecomprises a signal generating compound as described hereinaboveconjugated to a specific binding member of a specific binding pair, suchas carbazole or adamantane.

The various “signal-generating compounds” (labels) contemplated includechromagens, catalysts such as enzymes, luminescent compounds such asfluorescein and rhodamine, chemiluminescent compounds such asdioxetanes, acridiniums, phenanthridiniums and luminol, radioactiveelements and direct visual labels. Examples of enzymes include alkalinephosphatase, horseradish peroxidase, beta-galactosidase and the like.The selection of a particular label is not critical, but it must becapable of producing a signal either by itself or in conjunction withone or more additional substances.

“Solid phases” (“solid supports”) are known to those in the art andinclude the walls of wells of a reaction tray, test tubes, polystyrenebeads, magnetic or non-magnetic beads, nitrocellulose strips, membranes,microparticles such as latex particles, sheep (or other animal) redblood cells and Duracytes® (red blood cells “fixed” by pyruvic aldehydeand formaldehyde, available from Abbott Laboratories, Abbott Park, Ill.)and others. The “solid phase” is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and Duracytes® are all suitable examples. Suitable methods forimmobilizing peptides on solid phases include ionic, hydrophobic,covalent interactions and the like. A “solid phase,” as used herein,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. The solid phase can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid phase and which has the ability to immobilize the capturereagent through a specific binding reaction. The receptor moleculeenables the indirect binding of the capture reagent to a solid phasematerial before the performance of the assay or during the performanceof the assay. The solid phase thus can be a plastic, derivatizedplastic, magnetic or non-magnetic metal, glass or silicon surface of atest tube, microtiter well, sheet, bead, microparticle, chip, sheep (orother suitable animal's) red blood cells, Duracytes® and otherconfigurations known to those of ordinary skill in the art.

It is contemplated and within the scope of the present invention thatthe solid phase also can comprise any suitable porous material withsufficient porosity to allow access by detection antibodies and asuitable surface affinity to bind antigens. Microporous structuresgenerally are preferred, but materials with a gel structure in thehydrated state may be used as well. Such useful solid supports include,but are not limited to, nitrocellulose and nylon. It is contemplatedthat such porous solid supports described herein preferably are in theform of sheets of thickness from about 0.01 to 0.5 mm, preferably about0.1 mm. The pore size may vary within wide limits and preferably is fromabout 0.025 to 15 microns, especially from about 0.15 to 15 microns. Thesurface of such supports may be activated by chemical processes whichcause covalent linkage of the antigen or antibody to the support. Theirreversible binding of the antigen or antibody is obtained, however, ingeneral, by adsorption on the porous material by poorly understoodhydrophobic forces. Other suitable solid supports are known in the art.

Reagents.

The present invention provides reagents such as polynucleotide sequencesderived from a prostate tissue of interest and designated as PS108,polypeptides encoded thereby and antibodies specific for thesepolypeptides. The present invention also provides reagents such asoligonucleotide fragments derived from the disclosed polynucleotides andnucleic acid sequences complementary to these polynucleotides. Thepolynucleotides, polypeptides, or antibodies of the present inventionmay be used to provide information leading to the detecting, diagnosing,staging, monitoring, prognosticating, in vivo imaging, preventing ortreating of, or determining the predisposition to, diseases andconditions of the prostate, such as prostate cancer. The sequencesdisclosed herein represent unique polynucleotides which can be used inassays or for producing a specific profile of gene transcriptionactivity. Such assays are disclosed in European Patent Number 0373203B1and International Publication No. WO 95/11995, which are herebyincorporated by reference.

Selected PS108-derived polynucleotides can be used in the methodsdescribed herein for the detection of normal or altered gene expression.Such methods may employ PS108 polynucleotides or oligonucleotides,fragments or derivatives thereof, or nucleic acid sequencescomplementary thereto.

The polynucleotides disclosed herein, their complementary sequences, orfragments of either, can be used in assays to detect, amplify orquantify genes, nucleic acids, cDNAs or mRNAs relating to prostatetissue disease and conditions associated therewith. They also can beused to identify an entire or partial coding region of a PS108polypeptide. They further can be provided in individual containers inthe form of a kit for assays, or provided as individual compositions. Ifprovided in a kit for assays, other suitable reagents such as buffers,conjugates and the like may be included.

The polynucleotide may be in the form of RNA or DNA. Polynucleotides inthe form of DNA, cDNA, genomic DNA, nucleic acid analogs and syntheticDNA are within the scope of the present invention. The DNA may bedouble-stranded or single-stranded, and if single stranded, may be thecoding (sense) strand or non-coding (anti-sense) strand. The codingsequence which encodes the polypeptide may be identical to the codingsequence provided herein or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same polypeptide as the DNA provided herein.

This polynucleotide may include only the coding sequence for thepolypeptide, or the coding sequence for the polypeptide and anadditional coding sequence such as a leader or secretory sequence or aproprotein sequence, or the coding sequence for the polypeptide (andoptionally an additional coding sequence) and non-coding sequence, suchas a non-coding sequence 5′ and/or 3′ of the coding sequence for thepolypeptide.

In addition, the invention includes variant polynucleotides containingmodifications such as polynucleotide deletions, substitutions oradditions; and any polypeptide modification resulting from the variantpolynucleotide sequence. A polynucleotide of the present invention alsomay have a coding sequence which is a naturally occurring allelicvariant of the coding sequence provided herein.

In addition, the coding sequence for the polypeptide may be fused in thesame reading frame to a polynucleotide sequence which aids in expressionand secretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the polypeptide. The polynucleotides may alsoencode for a proprotein which is the protein plus additional 5′ aminoacid residues. A protein having a prosequence is a proprotein and may,in some cases, be an inactive form of the protein. Once the prosequenceis cleaved, an active protein remains. Thus, the polynucleotide of thepresent invention may encode for a protein, or for a protein having aprosequence, or for a protein having both a presequence (leadersequence) and a prosequence.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the polypeptide fused to the marker in thecase of a bacterial host, or, for example, the marker sequence may be ahemagglutinin (HA) tag when a mammalian host, e.g. a COS-7 cell line, isfused. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein. See, for example, I. Wilson et al., Cell 37:767(1984).

It is contemplated that polynucleotides will be considered to hybridizeto the sequences provided herein if there is at least 50%, preferably atleast 70%, and more preferably at least 90% identity between thepolynucleotide and the sequence.

The present invention also provides an antibody produced by using apurified PS108 polypeptide of which at least a portion of thepolypeptide is encoded by a PS108 polynucleotide selected from thepolynucleotides provided herein. These antibodies may be used in themethods provided herein for the detection of PS108 antigen in testsamples. The presence of PS108 antigen in the test samples is indicativeof the presence of a prostate disease or condition. The antibody alsomay be used for therapeutic purposes, for example, in neutralizing theactivity of PS108 polypeptide in conditions associated with altered orabnormal expression.

The present invention further relates to a PS108 polypeptide which hasthe deduced amino acid sequence as provided herein, as well asfragments, analogs and derivatives of such polypeptide. The polypeptideof the present invention may be a recombinant polypeptide, a naturalpurified polypeptide or a synthetic polypeptide. The fragment,derivative or analog of the PS108 polypeptide may be one in which one ormore of the amino acid residues is substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code; or it may be one in which one or more ofthe amino acid residues includes a substituent group; or it may be onein which the polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol); or it may be one in which the additional aminoacids are fused to the polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of thepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are within the scope of the present invention. The polypeptidesand polynucleotides of the present invention are provided preferably inan isolated form and preferably purified.

Thus, a polypeptide of the present invention may have an amino acidsequence that is identical to that of the naturally occurringpolypeptide or that is different by minor variations due to one or moreamino acid substitutions. The variation may be a “conservative change”typically in the range of about 1 to 5 amino acids, wherein thesubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine or threonine with serine.In contrast, variations may include nonconservative changes, e.g.,replacement of a glycine with a tryptophan. Similar minor variations mayalso include amino acid deletions or insertions, or both. Guidance indetermining which and how many amino acid residues may be substituted,inserted or deleted without changing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software (DNASTAR Inc., Madison Wis.).

Probes constructed according to the polynucleotide sequences of thepresent invention can be used in various assay methods to providevarious types of analysis. For example, such probes can be used influorescent in situ hybridization (FISH) technology to performchromosomal analysis, and used to identify cancer-specific structuralalterations in the chromosomes, such as deletions or translocations thatare visible from chromosome spreads or detectable using PCR-generatedand/or allele specific oligonucleotides probes, allele specificamplification or by direct sequencing. Probes also can be labeled withradioisotopes, directly- or indirectly-detectable haptens, orfluorescent molecules, and utilized for in situ hybridization studies toevaluate the mRNA expression of the gene comprising the polynucleotidein tissue specimens or cells.

This invention also provides teachings as to the production of thepolynucleotides and polypeptides provided herein.

Probe Assays

The sequences provided herein may be used to produce probes which can beused in assays for the detection of nucleic acids in test samples. Theprobes may be designed from conserved nucleotide regions of thepolynucleotides of interest or from non-conserved nucleotide regions ofthe polynucleotide of interest. The design of such probes foroptimization in assays is within the skill of the routineer. Generally,nucleic acid probes are developed from non-conserved or unique regionswhen maximum specificity is desired, and nucleic acid probes aredeveloped from conserved regions when assaying for nucleotide regionsthat are closely related to, for example, different members of amulti-gene family or in related species like mouse and man.

The polymerase chain reaction (PCR) is a technique for amplifying adesired nucleic acid sequence (target) contained in a nucleic acid ormixture thereof. In PCR, a pair of primers are employed in excess tohybridize to the complementary strands of the target nucleic acid. Theprimers are each extended by a polymerase using the target nucleic acidas a template. The extension products become target sequencesthemselves, following dissociation from the original target strand. Newprimers then are hybridized and extended by a polymerase, and the cycleis repeated to geometrically increase the number of target sequencemolecules. PCR is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202,which are incorporated herein by reference.

The Ligase Chain Reaction (LCR) is an alternate method for nucleic acidamplification. In LCR, probe pairs are used which include two primary(first and second) and two secondary (third and fourth) probes, all ofwhich are employed in molar excess to target The first probe hybridizesto a first segment of the target strand, and the second probe hybridizesto a second segment of the target strand, the first and second segmentsbeing contiguous so that the primary probes abut one another in 5′phosphate-3′ hydroxyl relationship, and so that a ligase can covalentlyfuse or ligate the two probes into a fused product. In addition, a third(secondary) probe can hybridize to a portion of the first probe and afourth (secondary) probe can hybridize to a portion of the second probein a similar abutting fashion. Of course, if the target is initiallydouble stranded, the secondary probes also will hybridize to the targetcomplement in the first instance. Once the ligated strand of primaryprobes is separated from the target strand, it will hybridize with thethird and fourth probes which can be ligated to form a complementary,secondary ligated product. It is important to realize that the ligatedproducts are functionally equivalent to either the target or itscomplement. By repeated cycles of hybridization and ligation,amplification of the target sequence is achieved. This technique isdescribed more completely in EP-A-320 308 to K. Backman published Jun.16, 1989 and EP-A-439 182 to K. Backman et al, published Jul. 31, 1991,both of which are incorporated herein by reference.

For amplification of mRNAs, it is within the scope of the presentinvention to reverse transcribe mRNA into cDNA followed by polymerasechain reaction (RT-PCR); or, to use a single enzyme for both steps asdescribed in U.S. Pat. No. 5,322,770, which is incorporated herein byreference; or reverse transcribe mRNA into cDNA followed by asymmetricgap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall etal., PCR Methods and Applications 4: 80-84 (1994), which also isincorporated herein by reference.

Other known amplification methods which can be utilized herein includebut are not limited to the so-called “NASBA” or “3SR” techniquedescribed by J. C. Guatelli et al., PNAS U.S.A. 87:1874-1878 (1990) andalso described by J. Compton, Nature 350 (No. 6313):91-92 (1991); Q-betaamplification as described in published European Patent Application(EPA) No. 4544610; strand displacement amplification (as described in G.T. Walker et al., Clin. Chem. 42:9-13 [1996]) and European PatentApplication No. 684315; and target mediated amplification, as describedin International Publication No. WO 93/22461.

Detection of PS108 may be accomplished using any suitable detectionmethod, including those detection methods which are currently well knownin the art, as well as detection strategies which may evolve later.Examples of the foregoing presently known detection methods are herebyincorporated herein by reference. See, for example, Caskey et al., U.S.Pat. No. 5,582,989, Gelfand et al., U.S. Pat. No. 5,210,015. Examples ofsuch detection methods include target amplification methods as well assignal amplification technologies. An example of presently knowndetection methods would include the nucleic acid amplificationtechnologies referred to as PCR, LCR, NASBA, SDA, RCR and TMA. See, forexample, Caskey et al., U.S. Pat. No. 5,582,989, Gelfand et al., U.S.Pat. No. 5,210,015. All of the foregoing are hereby incorporated byreference. Detection may also be accomplished using signal amplificationsuch as that disclosed in Snitman et al., U.S. Pat. No. 5,273,882. Whilethe amplification of target or signal is preferred at present, it iscontemplated and within the scope of the present invention thatultrasensitive detection methods which do not require amplification canbe utilized herein.

Detection, both amplified and non-amplified, may be (combined) carriedout using a variety of heterogeneous and homogeneous detection formats.Examples of heterogeneous detection formats are disclosed in Snitman etal., U.S. Pat. No. 5,273,882, Albarella et al in EP-84114441.9, Urdea etal., U.S. Pat. No. 5,124,246, Ullman et al. U.S. Pat. No. 5,185,243 andKourilsky et al., U.S. Pat. No. 4,581,333. All of the foregoing arehereby incorporated by reference. Examples of homogeneous detectionformats are. disclosed in, Caskey et al., U.S. Pat. No. 5,582,989,Gelfand et al., U.S. Pat. No. 5,210,015, which are incorporated hereinby reference. Also contemplated and within the scope of the presentinvention is the use of multiple probes in the hybridization assay,which use improves sensitivity and amplification of the PS108 signal.See, for example, Caskey et al., U.S. Pat. No. 5,582,989, Gelfand etal., U.S. Pat. No. 5,210,015, which are incorporated herein byreference.

In one embodiment, the present invention generally comprises the stepsof contacting a test sample suspected of containing a targetpolynucleotide sequence with amplification reaction reagents comprisingan amplification primer, and a detection probe that can hybridize withan internal region of the amplicon sequences. Probes and primersemployed according to the method provided herein are labeled withcapture and detection labels, wherein probes are labeled with one typeof label and primers are labeled with another type of label.Additionally, the primers and probes are selected such that the probesequence has a lower melt temperature than the primer sequences. Theamplification reagents, detection reagents and test sample are placedunder amplification conditions whereby, in the presence of targetsequence, copies of the target sequence (an amplicon) are produced. Inthe usual case, the amplicon is double stranded because primers areprovided to amplify a target sequence and its complementary strand. Thedouble stranded amplicon then is thermally denatured to produce singlestranded amplicon members. Upon formation of the single strandedamplicon members, the mixture is cooled to allow the formation ofcomplexes between the probes and single stranded amplicon members.

As the single stranded amplicon sequences and probe sequences arecooled, the probe sequences preferentially bind the single strandedamplicon members. This finding is counterintuitive given that the probesequences generally are selected to be shorter than the primer sequencesand therefore have a lower melt temperature than the primers.Accordingly, the melt temperature of the amplicon produced by theprimers should also have a higher melt temperature than the probes.Thus, as the mixture cools, the re-formation of the double strandedamplicon would be expected. As previously stated, however, this is notthe case. The probes are found to preferentially bind the singlestranded amplicon members. Moreover, this preference of probe/singlestranded amplicon binding exists even when the primer sequences areadded in excess of the probes.

After the probe/single stranded amplicon member hybrids are formed, theyare detected. Standard heterogeneous assay formats are suitable fordetecting the hybrids using the detection labels and capture labelspresent on the primers and probes. The hybrids can be bound to a solidphase reagent by virtue of the capture label and detected by virtue ofthe detection label. In cases where the detection label is directlydetectable, the presence of the hybrids on the solid phase can bedetected by causing the label to produce a detectable signal, ifnecessary, and detecting the signal. In cases where the label is notdirectly detectable, the captured hybrids can be contacted with aconjugate, which generally comprises a binding member attached to adirectly detectable label. The conjugate becomes bound to the complexesand the conjugate's presence on the complexes can be detected with thedirectly detectable label. Thus, the presence of the hybrids on thesolid phase reagent can be determined. Those skilled in the art willrecognize that wash steps may be employed to wash away unhybridizedamplicon or probe as well as unbound conjugate.

Although the target sequence is described as single stranded, it also iscontemplated to include the case where the target sequence is actuallydouble stranded but is merely separated from its complement prior tohybridization with the amplification primer sequences. In the case wherePCR is employed in this method, the ends of the target sequences areusually known. In cases where LCR or a modification thereof is employedin the preferred method, the entire target sequence is usually known.Typically, the target sequence is a nucleic acid sequence such as, forexample, RNA or DNA.

The method provided herein can be used in well-known amplificationreactions that include thermal cycle reaction mixtures, particularly inPCR and gap LCR (GLCR). Amplification reactions typically employ primersto repeatedly generate copies of a target nucleic acid sequence, whichtarget sequence is usually a small region of a much larger nucleic acidsequence. Primers are themselves nucleic acid sequences that arecomplementary to regions of a target sequence. Under amplificationconditions, these primers hybridize or bind to the complementary regionsof the target sequence. Copies of the target sequence typically aregenerated by the process of primer extension and/or ligation whichutilizes enzymes with polymerase or ligase activity, separately or incombination, to add nucleotides to the hybridized primers and/or ligateadjacent probe pairs. The nucleotides that are added to the primers orprobes, as monomers or preformed oligomers, are also complementary tothe target sequence. Once the primers or probes have been sufficientlyextended and/or ligated, they are separated from the target sequence,for example, by heating the reaction mixture to a “melt temperature”which is one in which complementary nucleic acid strands dissociate.Thus, a sequence complementary to the target sequence is formed.

A new amplification cycle then can take place to further amplify thenumber of target sequences by separating any double stranded sequences,allowing primers or probes to hybridize to their respective targets,extending and/or ligating the hybridized primers or probes andre-separating. The complementary sequences that are generated byamplification cycles can serve as templates for primer extension orfilling the gap of two probes to further amplify the number of targetsequences. Typically, a reaction mixture is cycled between 20 and 100times, more typically, a reaction mixture is cycled between 25 and 50times. The numbers of cycles can be determined by the routineer. In thismanner, multiple copies of the target sequence and its complementarysequence arc produced. Thus, primers initiate amplification of thetarget sequence when it is present under amplification conditions.

Generally, two primers which are complementary to a portion of a targetstrand and its complement are employed in PCR. For LCR, four probes, twoof which are complementary to a target sequence and two of which aresimilarly complementary to the target's complement, are generallyemployed. In addition to the primer sets and enzymes previouslymentioned, a nucleic acid amplification reaction mixture may alsocomprise other reagents which are well known and include but are notlimited to: enzyme cofactors such as manganese; magnesium; salts;nicotinamide adenine dinucleotide (NAD); and deoxynucleotidetriphosphates (dNTPs) such as, for example, deoxyadenine triphosphate,deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythyminetriphosphate.

While the amplification primers initiate amplification of the targetsequence, the detection (or hybridization) probe is not involved inamplification. Detection probes are generally nucleic acid sequences oruncharged nucleic acid analogs such as, for example, peptide nucleicacids which are disclosed in International Publication No. WO 92/20702;morpholino analogs which are described in U.S. Pat. Nos 5,185,444,5,034,506 and 5,142,047; and the like. Depending upon the type of labelcarried by the probe, the probe is employed to capture or detect theamplicon generated by the amplification reaction. The probe is notinvolved in amplification of the target sequence and therefore may haveto be rendered “non-extendible” in that additional dNTPs cannot be addedto the probe. In and of themselves, analogs usually are non-extendibleand nucleic acid probes can be rendered non-extendible by modifying the3′ end of the probe such that the hydroxyl group is no longer capable ofparticipating in elongation. For example, the 3′ end of the probe can befunctionalized with the capture or detection label to thereby consume orotherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl groupsimply can be cleaved, replaced or modified. U.S. patent applicationSer. No. 07/049,061 filed Apr. 19, 1993 and incorporated herein byreference describes modifications which can be used to render a probenon-extendible.

The ratio of primers to probes is not important. Thus, either the probesor primers can be added to the reaction mixture in excess whereby theconcentration of one would be greater than the concentration of theother. Alternatively, primers and probes can be employed in equivalentconcentrations. Preferably, however, the primers are added to thereaction mixture in excess of the probes. Thus, primer to probe ratiosof, for example, 5:1 and 20:1, are preferred.

While the length of the primers and probes can vary, the probe sequencesare selected such that they have a lower melt temperature than theprimer sequences. Hence, the primer sequences are generally longer thanthe probe sequences. Typically, the primer sequences are in the range ofbetween 20 and 50 nucleotides long, more typically in the range ofbetween 20 and 30 nucleotides long. The typical probe is in the range ofbetween 10 and 25 nucleotides long.

Various methods for synthesizing primers and probes are well known inthe art. Similarly, methods for attaching labels to primers or probesare also well known in the art. For example, it is a matter of routineto synthesize desired nucleic acid primers or probes using conventionalnucleotide phosphoramidite chemistry and instruments available fromApplied Biosystems, Inc., (Foster City, Calif.), DuPont (Wilmington,Del.), or Milligen (Bedford Mass.). Many methods have been described forlabeling oligonucleotides such as the primers or probes of the presentinvention. Enzo Biochemical (New York, N.Y.) and Clontech (Palo Alto,Calif.) both have described and commercialized probe labelingtechniques. For example, a primary amine can be attached to a 3′ oligoterminus using 3′-Amine-ON CPG™ (Clontech, Palo Alto, Calif.).Similarly, a primary amine can be attached to a 5′ oligo terminus usingAminomodifier II® (Clontech). The amines can be reacted to varioushaptens using conventional activation and linking chemistries. Inaddition, copending applications U.S. Ser. Nos. 625,566, filed Dec. 11,1990 and 630,908, filed Dec. 20, 1990, which are each incorporatedherein by reference, teach methods for labeling probes at their 5′ and3′ termini, respectively. International Publication Nos WO 92/10505,published Jun. 25, 1992, and WO 92/11388, published Jul. 9, 1992, teachmethods for labeling probes at their 5′ and 3′ ends, respectively.According to one known method for labeling an oligonucleotide, alabel-phosphoramidite reagent is prepared and used to add the label tothe oligonucleotide during its synthesis. See, for example, N. T. Thuonget al., Tet. Letters 29(46):5905-5908 (1988); or J. S. Cohen et al.,published U.S. patent application Ser. No. 07/246,688 (NTIS ORDER No.PAT-APPL-7-246,688) (1989). Preferably, probes are labeled at their 3′and 5′ ends.

A capture label is attached to the primers or probes and can be aspecific binding member which forms a binding pair with the solid phasereagent's specific binding member. It will be understood that the primeror probe itself may serve as the capture label. For example, in the casewhere a solid phase reagent's binding member is a nucleic acid sequence,it may be selected such that it binds a complementary portion of theprimer or probe to thereby immobilize the primer or probe to the solidphase. In cases where the probe itself serves as the binding member,those skilled in the art will recognize that the probe will contain asequence or “tail” that is not complementary to the single strandedamplicon members. In the case where the primer itself serves as thecapture label, at least a portion of the primer will be free tohybridize with a nucleic acid on a solid phase because the probe isselected such that it is not fully complementary to the primer sequence.

Generally, probe/single stranded amplicon member complexes can bedetected using techniques commonly employed to perform heterogeneousimmunoassays. Preferably, in this embodiment, detection is performedaccording to the protocols used by the commercially available AbbottLCx® instrumentation (Abbott Laboratories, Abbott Park, Ill.).

The primers and probes disclosed herein are useful in typical PCRassays, wherein the test sample is contacted with a pair of primers,amplification is performed, the hybridization probe is added, anddetection is performed.

Another method provided by the present invention comprises contacting atest sample with a plurality of polynucleotides, wherein at least onepolynucleotide is a PS108 molecule as described herein, hybridizing thetest sample with the plurality of polynucleotides and detectinghybridization complexes. Hybridization complexes are identified andquantitated to compile a profile which is indicative of prostate tissuedisease, such as prostate cancer. Expressed RNA sequences may further bedetected by reverse transcription and amplification of the DNA productby procedures well-known in the art, including polymerase chain reaction(PCR).

Drug Screening and Gene Therapy.

The present invention also encompasses the use of gene therapy methodsfor the introduction of anti-sense PS108 derived molecules, such aspolynucleotides or oligonucleotides of the present invention, intopatients with conditions associated with abnormal expression ofpolynucleotides related to a prostate tissue disease or conditionespecially prostate cancer. These molecules, including antisense RNA andDNA fragments and ribozymes, are designed to inhibit the translation ofPS108 mRNA, and may be used therapeutically in the treatment ofconditions associated with altered or abnormal expression of PS108polynucleotide.

Alternatively, the oligonucleotides described above can be delivered tocells by procedures known in the art such that the anti-sense RNA or DNAmay be expressed in vivo to inhibit production of a PS108 polypeptide inthe manner described above. Antisense constructs to a PS108polynucleotide, therefore, reverse the action of PS108 transcripts andmay be used for treating prostate tissue disease conditions, such asprostate cancer. These antisense constructs may also be used to treattumor metastases.

The present invention also provides a method of screening a plurality ofcompounds for specific binding to PS108 polypeptide(s), or any fragmentthereof, to identify at least one compound which specifically binds thePS108 polypeptide. Such a method comprises the steps of providing atleast one compound; combining the PS108 polypeptide with each compoundunder suitable conditions for a time sufficient to allow binding; anddetecting the PS108 polypeptide binding to each compound.

The polypeptide or peptide fragment employed in such a test may eitherbe free in solution, affixed to a solid support, borne on a cell surfaceor located intracellularly. One method of screening utilizes eukaryoticor prokaryotic host cells which are stably transfected with recombinantnucleic acids which can express the polypeptide or peptide fragment. Adrug, compound, or any other agent may be screened against suchtransfected cells in competitive binding assays. For example, theformation of complexes between a polypeptide and the agent being testedcan be measured in either viable or fixed cells.

The present invention thus provides methods of screening for drugs,compounds, or any other agent which can be used to treat diseasesassociated with PS108. These methods comprise contacting the agent witha polypeptide or fragment thereof and assaying for either the presenceof a complex between the agent and the polypeptide, or for the presenceof a complex between the polypeptide and the cell. In competitivebinding assays, the polypeptide typically is labeled. After suitableincubation, free (or uncomplexed) polypeptide or fragment thereof isseparated from that present in bound form, and the amount of free oruncomplexed label is used as a measure of the ability of the particularagent to bind to the polypeptide or to interfere with thepolypeptide/cell complex.

The present invention also encompasses the use of competitive screeningassays in which neutralizing antibodies capable of binding polypeptidespecifically compete with a test agent for binding to the polypeptide orfragment thereof. In this manner, the antibodies can be used to detectthe presence of any polypeptide in the test sample which shares one ormore antigenic determinants with a PS108 polypeptide as provided herein.

Another technique for screening provides high throughput screening forcompounds having suitable binding affinity to at least one polypeptideof PS108 disclosed herein. Briefly, large numbers of different smallpeptide test compounds are synthesized on a solid phase, such as plasticpins or some other surface. The peptide test compounds are reacted withpolypeptide and washed. Polypeptide thus bound to the solid phase isdetected by methods well-known in the art. Purified polypeptide can alsobe coated directly onto plates for use in the screening techniquesdescribed herein. In addition, non-neutralizing antibodies can be usedto capture the polypeptide and immobilize it on the solid support. See,for example, EP 84/03564, published on Sep. 13, 1984, which isincorporated herein by reference.

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of the small moleculesincluding agonists, antagonists, or inhibitors with which they interact.Such structural analogs can be used to design drugs which are moreactive or stable forms of the polypeptide or which enhance or interferewith the function of a polypeptide in vivo. J. Hodgson, Bio/Technology9:19-21 (1991), incorporated herein by reference.

For example, in one approach, the three-dimensional structure of apolypeptide, or of a polypeptide-inhibitor complex, is determined byx-ray crystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide may be gained by modeling basedon the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous polypeptide-likemolecules or to identify efficient inhibitors

Useful examples of rational drug design may include molecules which haveimproved activity or stability as shown by S. Braxton et al.,Biochemistry 31:7796-7801 (1992), or which act as inhibitors, agonists,or antagonists of native peptides as shown by S. B. P. Athauda et al., JBiochem. (Tokyo) 113 (6):742-746 (1993), incorporated herein byreference.

It also is possible to isolate a target-specific antibody selected by anassay as described hereinabove, and then to determine its crystalstructure. In principle this approach yields a pharmacophore upon whichsubsequent drug design can be based. It further is possible to bypassprotein crystallography altogether by generating anti-idiotypicantibodies (“anti-ids”) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-id is an analog of the original receptor. The anti-id then can beused to identify and isolate peptides from banks of chemically orbiologically produced peptides. The isolated peptides then can act asthe pharmacophore (that is, a prototype pharmaceutical drug).

A sufficient amount of a recombinant polypeptide of the presentinvention may be made available to perform analytical studies such asX-ray crystallography. In addition, knowledge of the polypeptide aminoacid sequence which is derivable from the nucleic acid sequence providedherein will provide guidance to those employing computer modelingtechniques in place of, or in addition to, x-ray crystallography.

Antibodies specific to a PS108 polypeptide (e.g., anti-PS108 antibodies)further may be used to inhibit the biological action of the polypeptideby binding to the polypeptide. In this manner, the antibodies may beused in therapy, for example, to treat prostate tissue diseasesincluding prostate cancer and its metastases.

Further, such antibodies can detect the presence or absence of a PS108polypeptide in a test sample and, therefore, are useful as diagnosticmarkers for the diagnosis of a prostate tissue disease or conditionespecially prostate cancer. Such antibodies may also function as adiagnostic marker for prostate tissue disease conditions, such asprostate cancer.

The present invention also is directed to antagonists and inhibitors ofthe polypeptides of the present invention. The antagonists andinhibitors are those which inhibit or eliminate the function of thepolypeptide. Thus, for example, an antagonist may bind to a polypeptideof the present invention and inhibit or eliminate its function. Theantagonist, for example, could be an antibody against the polypeptidewhich eliminates the activity of a PS108 polypeptide by binding a PS108polypeptide, or in some cases the antagonist may be an oligonucleotide.Examples of small molecule inhibitors include, but are not limited to,small peptides or peptide-like molecules.

The antagonists and inhibitors may be employed as a composition with apharmaceutically acceptable carrier including, but not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol andcombinations thereof. Administration of PS108 polypeptide inhibitors ispreferably systemic. The present invention also provides an antibodywhich inhibits the action of such a polypeptide.

Antisense technology can be used to reduce gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes for thepolypeptide of the present invention, is used to design an antisense RNAoligonucleotide of from 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription, thereby preventing transcription and theproduction of the PS108 polypeptide. For triple helix, see, for example,Lee et al, Nuc. Acids Res. 6:3073 (1979); Cooney et al, Science 241:456(1988); and Dervan et al, Science 251:1360 (1991) The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofa mRNA molecule into the PS108 polypeptide. For antisense, see, forexample, Okano, J. Neurochem. 56:560 (1991); and “Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression,” CRC Press, Boca Raton, Fla.(1988). Antisense oligonucleotides act with greater efficacy whenmodified to contain artificial internucleotide linkages which render themolecule resistant to nucleolytic cleavage. Such artificialinternucleotide linkages include, but are not limited to,methylphosphonate, phosphorothiolate and phosphoroamydateinternucleotide linkages.

Recombinant Technology.

The present invention provides host cells and expression vectorscomprising PS108 polynucleotides of the present invention and methodsfor the production of the polypeptide(s) they encode. Such methodscomprise culturing the host cells under conditions suitable for theexpression of the PS108 polynucleotide and recovering the PS108polypeptide from the cell culture.

The present invention also provides vectors which include PS108polynucleotides of the present invention, host cells which aregenetically engineered with vectors of the present invention and theproduction of polypeptides of the present invention by recombinanttechniques.

Host cells are genetically engineered (transfected, transduced ortransformed) with the vectors of this invention which may be cloningvectors or expression vectors. The vector may be in the form of aplasmid, a viral particle, a phage, etc. The engineered host cells canbe cultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transfected cells, or amplifying PS108gene(s). The culture conditions, such as temperature, pH and the like,are those previously used with the host cell selected for expression,and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, thepolynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular, vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virusand pseudorabies. However, any other plasmid or vector may be used solong as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted intoappropriate restriction endonuclease sites by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art. The DNA sequence in the expression vector isoperatively linked to an appropriate expression control sequence(s)(promoter) to direct mRNA synthesis. Representative examples of suchpromoters include, but are not limited to, the LTR or the SV40 promoter,the E. coli lac or trp, the phage lambda P sub L promoter and otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. The expression vector also contains aribosome binding site for translation initiation and transcriptionterminator. The vector may also include appropriate sequences foramplifying expression. In addition, the expression vectors preferablycontain a gene to provide a phenotypic trait for selection oftransfected host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transfect an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Salmonellatyphimurium; Streptomyces sp.; fungal cells, such as yeast; insectcells, such as Drosophila and Sf9; animal cells, such as CHO, COS orBowes melanoma; plant cells, etc. The selection of an appropriate hostis deemed to be within the scope of those skilled in the art from theteachings provided herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequencesincluding, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art and are commercially available. The following vectorsare provided by way of example. Bacterial: pINCY (Incyte PharmaceuticalsInc., Palo Alto, Calif.), pSPORT1 (Life Technologies, Gaithersburg,Md.), pQE70, pQE60, pQE-9 (Qiagen) pBs, phagescript, psiX174,pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene);pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic:pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL(Pharmacia). However, any other plasmid or vector may be used as long asit is replicable and viable in the host.

Plasmid pINCY is generally identical to the plasmid pSPORT1 (availablefrom Life Technologies, Gaithersburg, Md.) with the exception that ithas two modifications in the polylinker (multiple cloning site). Thesemodifications are (1) it lacks a HindIII restriction site and (2) itsEcoRI restriction site lies at a different location. pINCY is createdfrom pSPORT1 by cleaving pSPORT1 with both HindIII and EcoRI andreplacing the excised fragment of the polylinker with synthetic DNAfragments (SEQUENCE ID NO 17 and SEQUENCE ID NO 18). This replacementmay be made in any manner known to those of ordinary skill in the art.For example, the two nucleotide sequences, SEQUENCE ID NO 17 andSEQUENCE ID NO 18, may be generated synthetically with 5′ terminalphosphates, mixed together, and then ligated under standard conditionsfor performing staggered end ligations into the pSPORT1 plasmid cut withHindIII and EcoRI. Suitable host cells (such as E. coli DH5μ cells) thenare transfected with the ligated DNA and recombinant clones are selectedfor ampicillin resistance. Plasmid DNA then is prepared from individualclones and subjected to restriction enzyme analysis or DNA sequencing inorder to confirm the presence of insert sequences in the properorientation. Other cloning strategies known to the ordinary artisan alsomay be employed.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, SP6, T7, gpt, lambda P subR, P sub L and trp. Eukaryotic promoters include cytomegalovirus (CMV)immediate early, herpes simplex virus (HSV) thymidine kinase, early andlate SV40, LTRs from retroviruses and mouse metallothionein-I. Selectionof the appropriate vector and promoter is well within the level ofordinary skill in the art.

In a further embodiment, the present invention provides host cellscontaining the above-described construct. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (L. Davis et al., “Basic Methods inMolecular Biology,” 2nd edition, Appleton and Lang, ParamountPublishing, East Norwalk, Conn. (1994)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Recombinant proteins can be expressed in mammalian cells, yeast,bacteria, or other cells, under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (Cold SpringHarbor, N.Y., 1989), which is hereby incorporated by reference.

Transcription of a DNA encoding the polypeptide(s) of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication originand adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transfection of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransfection include E. coli, Bacillus subtilis, Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces andStaphylococcus, although others may also be employed as a routine matterof choice.

Useful expression vectors for bacterial use comprise a selectable markerand bacterial origin of replication derived from plasmids comprisinggenetic elements of the well-known cloning vector pBR322 (ATCC 37017).Other vectors include but are not limited to PKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.).These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transfection of a suitable host and growth of the host to anappropriate cell density, the selected promoter is derepressed byappropriate means (e.g., temperature shift or chemical induction), andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in expression of proteins can be disrupted byany convenient method including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell-known to the ordinary artisan.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, such as the C 127, HEK-293, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements. Representative, useful vectors include pRc/CMV andpcDNA3 (available from Invitrogen, San Diego, Calif.).

PS108 polypeptides are recovered and purified from recombinant cellcultures by known methods including affinity chromatography, ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, hydroxyapatite chromatography or lectinchromatography. It is preferred to have low concentrations(approximately 0.1-5 mM) of calcium ion present during purification(Price, et al., J. Biol. Chem. 244:917 (1969)). Protein refolding stepscan be used, as necessary, in completing configuration of thepolypeptide. Finally, high performance liquid chromatography (HPLC) canbe employed for final purification steps.

Thus, polypeptides of the present invention may be naturally purifiedproducts expressed from a high expressing cell line, or a product ofchemical synthetic procedures, or produced by recombinant techniquesfrom a prokaryotic or eukaryotic host (for example, by bacterial, yeast,higher plant, insect and mammalian cells in culture). Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated with mammalian or othereukaryotic carbohydrates or may be non-glycosylated. The polypeptides ofthe invention may also include an initial methionine amino acid residue.

The starting plasmids can be constructed from available plasmids inaccord with published, known procedures. In addition, equivalentplasmids to those described are known in the art and will be apparent toone of ordinary skill in the art.

The following is the general procedure for the isolation and analysis ofcDNA clones. In a particular embodiment disclosed herein, mRNA wasisolated from prostate tissue and used to generate the cDNA library.Prostate tissue was obtained from patients by surgical resection and wasclassified as tumor or non-tumor tissue by a pathologist.

The cDNA inserts from random isolates of the prostate tissue librarieswere sequenced in part, analyzed in detail as set forth in the Examples,and are disclosed in the Sequence Listing as SEQUENCE ID NOS 1-14. Alsoanalyzed in detail as set forth in the Examples, and disclosed in theSequence Listing, is the full-length sequence of clone 1711346 (referredto as clone 1711346IH, (SEQUENCE ID NO 15)). The consensus sequence ofthese inserts is presented as SEQUENCE ID NO 16. These polynucleotidesmay contain an entire open reading frame with or without associatedregulatory sequences for a particular gene, or they may encode only aportion of the gene of interest. This is attributed to the fact thatmany genes arc several hundred and sometimes several thousand bases inlength and, with current technology, cannot be cloned in their entiretybecause of vector limitations, incomplete reverse transcription of thefirst strand, or incomplete replication of the second strand.Contiguous, secondary clones containing additional nucleotide sequencesmay be obtained using a variety of methods known to those of skill inthe art.

Methods for DNA sequencing arc well known in the art. Conventionalenzymatic methods employ DNA polymerase, Klenow fragment, Sequenase (USBiochemical Corp, Cleveland, Ohio) or Taq polymerase to extend DNAchains from an oligonucleotide primer annealed to the DNA template ofinterest. Methods have been developed for the use of bothsingle-stranded and double-stranded templates. The chain terminationreaction products may be electrophoresed on urea/polyacrylamide gels anddetected either by autoradiography (for radionucleotide labeledprecursors) or by fluorescence (for fluorescent-labeled precursors).Recent improvements in mechanized reaction preparation, sequencing andanalysis using the fluorescent detection method have permitted expansionin the number of sequences that can be determined per day using machinessuch as the Applied Biosystems 377 DNA Sequencers (Applied Biosystems,Foster City, Calif.).

The reading frame of the nucleotide sequence can be ascertained byseveral types of analyses. First, reading frames contained within thecoding sequence can be analyzed for the presence of start codon ATG andstop codons TGA, TAA or TAG. Typically, one reading frame will continuethroughout the major portion of a cDNA sequence while other readingframes tend to contain numerous stop codons. In such cases, readingframe determination is straightforward. In other more difficult cases,further analysis is required.

Algorithms have been created to analyze the occurrence of individualnucleotide bases at each putative codon triplet. See, for example J. W.Fickett, Nuc. Acids Res. 10:5303 (1982). Coding DNA for particularorganisms (bacteria, plants and animals) tends to contain certainnucleotides within certain triplet periodicities, such as a significantpreference for pyrimidines in the third codon position. Thesepreferences have been incorporated into widely available software whichcan be used to determine coding potential (and frame) of a given stretchof DNA. The algorithm-derived information combined with start/stop codoninformation can be used to determine proper frame with a high degree ofcertainty. This, in turn, readily permits cloning of the sequence in thecorrect reading frame into appropriate expression vectors.

The nucleic acid sequences disclosed herein may be joined to a varietyof other polynucleotide sequences and vectors of interest by means ofwell-established recombinant DNA techniques. See J. Sambrook et al.,supra. Vectors of interest include cloning vectors, such as plasmids,cosmids, phage derivatives, phagemids, as well as sequencing,replication and expression vectors, and the like. In general, suchvectors contain an origin of replication functional in at least oneorganism, convenient restriction endonuclease digestion sites andselectable markers appropriate for particular host cells. The vectorscan be transferred by a variety of means known to those of skill in theart into suitable host cells which then produce the desired DNA, RNA orpolypeptides.

Occasionally, sequencing or random reverse transcription errors willmask the presence of the appropriate open reading frame or regulatoryelement. In such cases, it is possible to determine the correct readingframe by attempting to express the polypeptide and determining the aminoacid sequence by standard peptide mapping and sequencing techniques.See, F. M. Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y. (1989). Additionally, the actual readingframe of a given nucleotide sequence may be determined by transfectionof host cells with vectors containing all three potential readingframes. Only those cells with the nucleotide sequence in the correctreading frame will produce a peptide of the predicted length.

The nucleotide sequences provided herein have been prepared by current,state-of-the-art, automated methods and, as such, may containunidentified nucleotides. These will not present a problem to thoseskilled in the art who wish to practice the invention. Several methodsemploying standard recombinant techniques, described in J. Sambrook(supra) or periodic updates thereof, may be used to complete the missingsequence information. The same techniques used for obtaining a fulllength sequence, as described herein, may be used to obtain nucleotidesequences.

Expression of a particular cDNA may be accomplished by subcloning thecDNA into an appropriate expression vector and transfecting this vectorinto an appropriate expression host. The cloning vector used for thegeneration of the prostate tissue cDNA library can be used fortranscribing mRNA of a particular cDNA and contains a promoter forbeta-galactosidase, an amino-terminal met and the subsequent seven aminoacid residues of beta-galactosidase. Immediately following these eightresidues is an engineered bacteriophage promoter useful for artificialpriming and transcription, as well as a number of unique restrictionsites, including EcoRI, for cloning. The vector can be transfected intoan appropriate host strain of E. coli.

Induction of the isolated bacterial strain with isopropylthiogalactoside(IPTG) using standard methods will produce a fusion protein whichcontains the first seven residues of beta-galactosidase, about 15residues of linker and the peptide encoded within the cDNA. Since cDNAclone inserts are generated by an essentially random process, there isone chance in three that the included cDNA will lie in the correct framefor proper translation. If the cDNA is not in the proper reading frame,the correct frame can be obtained by deletion or insertion of anappropriate number of bases by well known methods including in vitromutagenesis, digestion with exonuclease III or mung bean nuclease, oroligonucleotide linker inclusion.

The cDNA can be shuttled into other vectors known to be useful forexpression of protein in specific hosts. Oligonuclcotide primerscontaining cloning sites and segments of DNA sufficient to hybridize tostretches at both ends of the target cDNA can be synthesized chemicallyby standard methods. These primers can then be used to amplify thedesired gene segments by PCR. The resulting new gene segments can bedigested with appropriate restriction enzymes under standard conditionsand isolated by gel electrophoresis. Alternately, similar gene segmentscan be produced by digestion of the cDNA with appropriate restrictionenzymes and filling in the missing gene segments with chemicallysynthesized oligonucleotides. Segments of the coding sequence from morethan one gene can be ligated together and cloned in appropriate vectorsto optimize expression of recombinant sequence.

Suitable expression hosts for such chimeric molecules include, but arenot limited to, mammalian cells, such as Chinese Hamster Ovary (CHO) andhuman embryonic kidney (HEK) 293 cells, insect cells, such as Sf9 cells,yeast cells, such as Saccharomyces cerevisiae and bacteria, such as E.coli. For each of these cell systems, a useful expression vector mayalso include an origin of replication to allow propagation in bacteriaand a selectable marker such as the beta-lactamase antibiotic resistancegene to allow selection in bacteria. In addition, the vectors mayinclude a second selectable marker, such as the neomycinphosphotransferase gene, to allow selection in transfected eukaryotichost cells. Vectors for use in eukaryotic expression hosts may requirethe addition of 3′ poly A tail if the sequence of interest lacks poly A.

Additionally, the vector may contain promoters or enhancers whichincrease gene expression. Such promoters are host specific and include,but are not limited to, MMTV, SV40, or metallothionine promoters for CHOcells; trp, lac, tac or T7 promoters for bacterial hosts; or alphafactor, alcohol oxidase or PGH promoters for yeast. Adenoviral vectorswith or without transcription enhancers, such as the Rous sarcoma virus(RSV) enhancer, may be used to drive protein expression in mammaliancell lines. Once homogeneous cultures of recombinant cells are obtained,large quantities of recombinantly produced protein can be recovered fromthe conditioned medium and analyzed using chromatographic methods wellknown in the art. An alternative method for the production of largeamounts of secreted protein involves the transfection of mammalianembryos and the recovery of the recombinant protein from milk producedby transgenic cows, goats, sheep, etc. Polypeptides and closely relatedmolecules may be expressed recombinantly in such a way as to facilitateprotein purification. One approach involves expression of a chimericprotein which includes one or more additional polypeptide domains notnaturally present on human polypeptides. Such purification-facilitatingdomains include, but are not limited to, metal-chelating peptides suchas histidine-tryptophan domains that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle, Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase fromInvitrogen (San Diego, Calif.) between the polypeptide sequence and thepurification domain may be useful for recovering the polypeptide.

Immunoassays.

PS108 polypeptides, including fragments, derivatives, and analogsthereof, or cells expressing such polypeptides, can be utilized in avariety of assays, many of which are described herein, for the detectionof antibodies to prostate tissue. They also can be used as immunogens toproduce antibodies. These antibodies can be, for example, polyclonal ormonoclonal antibodies, chimeric, single chain and humanized antibodies,as well as Fab fragments, or the product of an Fab expression library.Various procedures known in the art may be used for the production ofsuch antibodies and fragments.

For example, antibodies generated against a polypeptide comprising asequence of the present invention can be obtained by direct injection ofthe polypeptide into an animal or by administering the polypeptide to ananimal such as a mouse, rabbit, goat or human. A mouse, rabbit or goatis preferred. The polypeptide is selected from the group consisting ofSEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38, SEQUENCE ID NO39, and fragments thereof. The antibody so obtained then will bind thepolypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptide can be used to generate antibodies that bindthe native polypeptide. Such antibodies then can be used to isolate thepolypeptide from test samples such as tissue suspected of containingthat polypeptide. For preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples include the hybridoma technique asdescribed by Kohler and Milstein, Nature 256:495-497 (1975), the triomatechnique, the human B-cell hybridoma technique as described by Kozboret al, Immun. Today 4:72 (1983) and the EBV-hybridoma technique toproduce human monoclonal antibodies as described by Cole et al., inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc, New York,N.Y., pp. 77-96 (1985). Techniques described for the production ofsingle chain antibodies can be adapted to produce single chainantibodies to immunogenic polypeptide products of this invention. See,for example, U.S. Pat. No. 4,946,778, which is incorporated herein byreference.

Various assay formats may utilize the antibodies of the presentinvention, including “sandwich” immunoassays and probe assays. Forexample, the antibodies of the present invention, or fragments thereof,can be employed in various assay systems to determine the presence, ifany, of PS108 antigen in a test sample. For example, in a first assayformat, a polyclonal or monoclonal antibody or fragment thereof, or acombination of these antibodies, which has been coated on a solid phase,is contacted with a test sample, to form a first mixture. This firstmixture is incubated for a time and under conditions sufficient to formantigen/antibody complexes. Then, an indicator reagent comprising amonoclonal or a polyclonal antibody or a fragment thereof, or acombination of these antibodies, to which a signal generating compoundhas been attached, is contacted with the antigen/antibody complexes toform a second mixture. This second mixture then is incubated for a timeand under conditions sufficient to form antibody/antigen/antibodycomplexes. The presence of PS108 antigen in the test sample and capturedon the solid phase, if any, is determined by detecting the measurablesignal generated by the signal generating compound. The amount of PS108antigen present in the test sample is proportional to the signalgenerated.

In an alternative assay format, a mixture is formed by contacting: (1) apolyclonal antibody, monoclonal antibody, or fragment thereof, whichspecifically binds to PS108 antigen, or a combination of such antibodiesbound to a solid support; (2) the test sample; and (3) an indicatorreagent comprising a monoclonal antibody, polyclonal antibody, orfragment thereof, which specifically binds to a different PS108 antigen(or a combination of these antibodies) to which a signal generatingcompound is attached. This mixture is incubated for a time and underconditions sufficient to form antibody/antigen/antibody complexes. Thepresence, if any, of PS108 antigen present in the test sample andcaptured on the solid phase is determined by detecting the measurablesignal generated by the signal generating compound. The amount of PS108antigen present in the test sample is proportional to the signalgenerated.

In another assay format, one or a combination of at least two monoclonalantibodies of the invention can be employed as a competitive probe forthe detection of antibodies to PS108 antigen. For example, PS108polypeptides such as the recombinant antigens disclosed herein, eitheralone or in combination, are coated on a solid phase. A test samplesuspected of containing antibody to PS108 antigen then is incubated withan indicator reagent comprising a signal generating compound and atleast one monoclonal antibody of the invention for a time and underconditions sufficient to form antigen/antibody complexes of either thetest sample and indicator reagent bound to the solid phase or theindicator reagent bound to the solid phase. The reduction in binding ofthe monoclonal antibody to the solid phase can be quantitativelymeasured.

In yet another detection method, each of the monoclonal or polyclonalantibodies of the present invention can be employed in the detection ofPS108 antigens in tissue sections, as well as in cells, byimmunohistochemical analysis. The tissue sections can be cut from eitherfrozen or chemically fixed samples of tissue. If the antigens are to bedetected in cells, the cells can be isolated from blood, urine, breastaspirates, or other bodily fluids. The cells may be obtained by biopsy,either surgical or by needle. The cells can be isolated bycentrifugation or magnetic attraction after labeling with magneticparticles or ferrofluids so as to enrich a particular fraction of cellsfor staining with the antibodies of the present invention. Cytochemicalanalysis wherein these antibodies are labeled directly (with, forexample, fluorescein, colloidal gold, horseradish peroxidase, alkalinephosphatase, etc.) or are labeled by using secondary labeledanti-species antibodies (with various labels as exemplified herein) totrack the histopathology of disease also are within the scope of thepresent invention.

In addition, these monoclonal antibodies can be bound to matricessimilar to CNBr-activated Sepharose and used for the affinitypurification of specific PS108 polypeptides from cell cultures orbiological tissues such as to purify recombinant and native PS108proteins.

The monoclonal antibodies of the invention also can be used for thegeneration of chimeric antibodies for therapeutic use, or other similarapplications.

The monoclonal antibodies or fragments thereof can be providedindividually to detect PS108 antigens. Combinations of the monoclonalantibodies (and fragments thereof) provided herein also may be usedtogether as components in a mixture or “cocktail” of at least one PS108antibody of the invention, along with antibodies which specifically bindto other PS108 regions, each antibody having different bindingspecificities. Thus, this cocktail can include the monoclonal antibodiesof the invention which are directed to PS108 polypeptides disclosedherein and other monoclonal antibodies specific to other antigenicdeterminants of PS108 antigens or other related proteins.

The polyclonal antibody or fragment thereof which can be used in theassay formats should specifically bind to a PS108 polypeptide or otherPS108 polypeptides additionally used in the assay. The polyclonalantibody used preferably is of mammalian origin such as, human, goat,rabbit or sheep polyclonal antibody which binds PS108 polypeptide. Mostpreferably, the polyclonal antibody is of rabbit origin. The polyclonalantibodies used in the assays can be used either alone or as a cocktailof polyclonal antibodies. Since the cocktails used in the assay formatsare comprised of either monoclonal antibodies or polyclonal antibodieshaving different binding specificity to PS108 polypeptides, they areuseful for the detecting, diagnosing, staging, monitoring,prognosticating, in vivo imaging, preventing or treating, or determiningthe predisposition to, diseases and conditions of the prostate, such asprostate cancer.

It is contemplated and within the scope of the present invention thatPS108 antigen may be detectable in assays by use of a recombinantantigen as well as by use of a synthetic peptide or purified peptide,which peptide comprises an amino acid sequence of PS108. The amino acidsequence of such a polypeptide is selected from the group consisting ofSEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38, SEQUENCE ID NO39, and fragments thereof. It also is within the scope of the presentinvention that different synthetic, recombinant or purified peptides,identifying different epitopes of PS108, can be used in combination inan assay for the detecting, diagnosing, staging, monitoring,prognosticating, in vivo imaging, preventing or treating, or determiningthe predisposition to diseases and conditions of the prostate, such asprostate cancer. In this case, all of these peptides can be coated ontoone solid phase; or each separate peptide may be coated onto separatesolid phases, such as microparticles, and then combined to form amixture of peptides which can be later used in assays. Furthermore, itis contemplated that multiple peptides which define epitopes fromdifferent antigens may be used for the detection, diagnosis, staging,monitoring, prognosis, prevention or treatment of, or determining thepredisposition to, diseases and conditions of the prostate, such asprostate cancer. Peptides coated on solid phases or labeled withdetectable labels are then allowed to compete with those present in apatient sample (if any) for a limited amount of antibody. A reduction inbinding of the synthetic, recombinant, or purified peptides to theantibody (or antibodies) is an indication of the presence of PS108antigen in the patient sample. The presence of PS108 antigen indicatesthe presence of prostate tissue disease, especially prostate cancer, inthe patient. Variations of assay formats are known to those of ordinaryskill in the art and many are discussed herein below.

In another assay format, the presence of anti-PS108 antibody and/orPS108 antigen can be detected in a simultaneous assay, as follows. Atest sample is simultaneously contacted with a capture reagent of afirst analyte, wherein said capture reagent comprises a first bindingmember specific for a first analyte attached to a solid phase and acapture reagent for a second analyte, wherein said capture reagentcomprises a first binding member for a second analyte attached to asecond solid phase, to thereby form a mixture. This mixture is incubatedfor a time and under conditions sufficient to form capture reagent/firstanalyte and capture reagent/second analyte complexes. These so-formedcomplexes then are contacted with an indicator reagent comprising amember of a binding pair specific for the first analyte labeled with asignal generating compound and an indicator reagent comprising a memberof a binding pair specific for the second analyte labeled with a signalgenerating compound to form a second mixture. This second mixture isincubated for a time and under conditions sufficient to form capturereagent/first analyte/indicator reagent complexes and capturereagent/second analyte/indicator reagent complexes. The presence of oneor more analytes is determined by detecting a signal generated inconnection with the complexes formed on either or both solid phases asan indication of the presence of one or more analytes in the testsample. In this assay format, recombinant antigens derived from theexpression systems disclosed herein may be utilized, as well asmonoclonal antibodies produced from the proteins derived from theexpression systems as disclosed herein. For example, in this assaysystem, PS108 antigen can be the first analyte. Such assay systems aredescribed in greater detail in EP Publication No. 0473065.

In yet other assay formats, the polypeptides disclosed herein may beutilized to detect the presence of antibody against PS108 antigen intest samples. For example, a test sample is incubated with a solid phaseto which at least one polypeptide such as a recombinant protein orsynthetic peptide has been attached. The polypeptide is selected fromthe group consisting of SEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCEID NO 38, SEQUENCE ID NO 39, and fragments thereof. These are reactedfor a time and under conditions sufficient to form antigen/antibodycomplexes. Following incubation, the antigen/antibody complex isdetected. Indicator reagents may be used to facilitate detection,depending upon the assay system chosen. In another assay format, a testsample is contacted with a solid phase to which a recombinant proteinproduced as described herein is attached, and also is contacted with amonoclonal or polyclonal antibody specific for the protein, whichpreferably has been labeled with an indicator reagent. After incubationfor a time and under conditions sufficient for antibody/antigencomplexes to form, the solid phase is separated from the free phase, andthe label is detected in either the solid or free phase as an indicationof the presence of antibody against PS108 antigen. Other assay formatsutilizing the recombinant antigens disclosed herein are contemplated.These include contacting a test sample with a solid phase to which atleast one antigen from a first source has been attached, incubating thesolid phase and test sample for a time and under conditions sufficientto form antigen/antibody complexes, and then contacting the solid phasewith a labeled antigen, which antigen is derived from a second sourcedifferent from the first source. For example, a recombinant proteinderived from a first source such as E. coli is used as a capture antigenon a solid phase, a test sample is added to the so-prepared solid phase,and following standard incubation and washing steps as deemed orrequired, a recombinant protein derived from a different source (i.e.,non-E. coli) is utilized as a part of an indicator reagent whichsubsequently is detected. Likewise, combinations of a recombinantantigen on a solid phase and synthetic peptide in the indicator phasealso are possible. Any assay format which utilizes an antigen specificfor PS108 produced or derived from a first source as the capture antigenand an antigen specific for PS108 from a different second source iscontemplated. Thus, various combinations of recombinant antigens, aswell as the use of synthetic peptides, purified proteins and the like,are within the scope of this invention. Assays such as this and othersare described in U.S. Pat. No. 5,254,458, which enjoys common ownershipand is incorporated herein by reference.

Other embodiments which utilize various other solid phases also arecontemplated and are within the scope of this invention. For example,ion capture procedures for immobilizing an immobilizable reactioncomplex with a negatively charged polymer (described in EP publication0326100 and EP publication No. 0406473), can be employed according tothe present invention to effect a fast solution-phase immunochemicalreaction. An immobilizable immune complex is separated from the rest ofthe reaction mixture by ionic interactions between the negativelycharged poly-anion/immune complex and the previously treated, positivelycharged porous matrix and detected by using various signal generatingsystems previously described, including those described inchemiluminescent signal measurements as described in EPO Publication No.0 273,115.

Also, the methods of the present invention can be adapted for use insystems which utilize microparticle technology including automated andsemi-automated systems wherein the solid phase comprises a microparticle(magnetic or non-magnetic). Such systems include those described in, forexample, published EPO applications Nos. EP 0 425 633 and EP 0 424 634,respectively.

The use of scanning probe microscopy (SPM) for immunoassays also is atechnology to which the monoclonal antibodies of the present inventionare easily adaptable. In scanning probe microscopy, particularly inatomic force microscopy, the capture phase, for example, at least one ofthe monoclonal antibodies of the invention, is adhered to a solid phaseand a scanning probe microscope is utilized to detect antigen/antibodycomplexes which may be present on the surface of the solid phase. Theuse of scanning tunneling microscopy eliminates the need for labelswhich normally must be utilized in many immunoassay systems to detectantigen/antibody complexes. The use of SPM to monitor specific bindingreactions can occur in many ways. In one embodiment, one member of aspecific binding partner (analyte specific substance which is themonoclonal antibody of the invention) is attached to a surface suitablefor scanning. The attachment of the analyte specific substance may be byadsorption to a lest piece which comprises a solid phase of a plastic ormetal surface, following methods known to those of ordinary skill in theart. Or, covalent attachment of a specific binding partner (analytespecific substance) to a test piece which test piece comprises a solidphase of derivatized plastic, metal, silicon, or glass may be utilized.Covalent attachment methods are known to those skilled in the art andinclude a variety of means to irreversibly link specific bindingpartners to the test piece. If the test piece is silicon or glass, thesurface must be activated prior to attaching the specific bindingpartner. Also, polyelectrolyte interactions may be used to immobilize aspecific binding partner on a surface of a test piece by usingtechniques and chemistries. The preferred method of attachment is bycovalent means. Following attachment of a specific binding member, thesurface may be further treated with materials such as serum, proteins,or other blocking agents to minimize non-specific binding. The surfacealso may be scanned either at the site of manufacture or point of use toverify its suitability for assay purposes. The scanning process is notanticipated to alter the specific binding properties of the test piece.

While the present invention discloses the preference for the use ofsolid phases, it is contemplated that the reagents such as antibodies,proteins and peptides of the present invention can be utilized innon-solid phase assay systems. These assay systems are known to thoseskilled in the art, and are considered to be within the scope of thepresent invention.

It is contemplated that the reagent employed for the assay can beprovided in the form of a test kit with one or more containers such asvials or bottles, with each container containing a separate reagent suchas a probe, primer, monoclonal antibody or a cocktail of monoclonalantibodies, or a polypeptide (e.g. recombinantly, synthetically producedor purified) employed in the assay. The polypeptide is selected from thegroup consisting of SEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO38, SEQUENCE ID NO 39, and fragments thereof. Other components such asbuffers, controls and the like, known to those of ordinary skill in art,may be included in such test kits. It also is contemplated to providetest kits which have means for collecting test samples comprisingaccessible body fluids, e.g., blood, urine, saliva and stool. Such toolsuseful for collection (“collection materials”) include lancets andabsorbent paper or cloth for collecting and stabilizing blood; swabs forcollecting and stabilizing saliva; cups for collecting and stabilizingurine or stool samples. Collection materials, papers, cloths, swabs,cups and the like, may optionally be treated to avoid denaturation orirreversible adsorption of the sample. The collection materials also maybe treated with or contain preservatives, stabilizers or antimicrobialagents to help maintain the integrity of the specimens. Test kitsdesigned for the collection, stabilization and preservation of testspecimens obtained by surgery or needle biopsy are also useful. It iscontemplated that all kits may be configured in two components which canbe provided separately; one component for collection and transport ofthe specimen and the other component for the analysis of the specimen.The collection component, for example, can be provided to the openmarket user while the components for analysis can be provided to otherssuch as laboratory personnel for determination of the presence, absenceor amount of analyte. Further, kits for the collection, stabilizationand preservation of test specimens may be configured for use byuntrained personnel and may be available in the open market for use athome with subsequent transportation to a laboratory for analysis of thetest sample.

In Vivo Antibody Use.

Antibodies of the present invention can be used in vivo; that is, theycan be injected into patients suspected of having or having diseases ofthe prostate for diagnostic or therapeutic uses. The use of antibodiesfor in vivo diagnosis is well known in the art. Sumerdon et al, Nucl.Med. Biol, 17, 247-254 (1990) have described an optimizedantibody-chelator for the radioimmunoscintographic imaging ofcarcinoembryonic antigen (CEA) expressing tumors using Indium-111 as thelabel. Griffin et al, J Clin Onc, 9, 631-640 (1991) have described theuse of this agent in detecting tumors in patients suspected of havingrecurrent colorectal cancer. The use of similar agents with paramagneticions as labels for magnetic resonance imaging is know in the art (R. B.Lauffer, Magnetic Resonance in Medicine, 22, 339-342 (1991). It isanticipated that antibodies directed against PS108 antigen can beinjected into patients suspected of having a disease of the prostatesuch as prostate cancer for the purpose of diagnosing or staging thedisease status of the patient. The label used will depend on the imagingmodality chosen. Radioactive labels such as Indium-111, Technetium-99m,or Iodine-131 can be used for planar scans or single photon emissioncomputed tomography (SPECT). Positron emitting labels such asFluorine-19 can also be used for positron emission tomography (PET). ForMRI, paramagnetic ions such as Gadolinium (III) or Manganese (II) can beused. Localization of the label within the prostate or external to theprostate may allow determination of spread of the disease. The amount oflabel within the prostate may allow determination of the presence orabsence of cancer of the prostate.

For patients known to have a disease of the prostate, injection of anantibody directed against PS108 antigen may halve therapeutic benefit.The antibody may exert its effect without the use of attached agents bybinding to PS108 antigen expressed on or in the tissue or organ.Alternatively, the antibody may be conjugated to cytotoxic agents suchas drugs, toxins, or radionuclides to enhance its therapeutic effect.Garnett and Baldwin, Cancer Research, 46, 2407-2412 (1986) havedescribed the preparation of a drug-monoclonal antibody conjugate.Pastan et al, Cell, 47, 641-648 (1986) have reviewed the use of toxinsconjugated to monoclonal antibodies for the therapy of various cancers.Goodwin and Meares, Cancer Supplement, 80, 2675-2680 (1997) havedescribed the use of Yttrium-90 labeled monoclonal antibodies in variousstrategies to maximize the dose to tumor while limiting normal tissuetoxicity. Other known cytotoxic radionuclides include Copper-67,Iodine-131, and Rhenium-186 all of which can be used to label monoclonalantibodies directed against PS108 antigen for the treatment of cancer ofthe prostate.

E. coli bacteria (clone 1711346) was deposited on Jan. 14, 1997 with theAmerican Type Culture Collection (A.T.C.C.), 12301 Parklawn Drive,Rockville, Md. 20852. The deposit was made under the terms of theBudapest Treaty and will be maintained for a period of thirty (30) yearsfrom the date of deposit, or for five (5) years after the last requestfor the deposit, or for the enforceable period of the U.S. patent,whichever is longer. The deposit and any other deposited materialdescribed herein are provided for convenience only, and are not requiredto practice the present invention in view of the teachings providedherein. The cDNA sequence in all of the deposited material isincorporated herein by reference. Clone 1711346 was accorded A.T.C.C.Deposit No. 98276.

The present invention will now be described by way of examples, whichare meant to illustrate, but not to limit, the scope of the presentinvention.

EXAMPLES Example 1 Identification of Prostate Tissue Library PS108Gene-Specific Clones

A. Library Comparison of Expressed Sequence Tags (EST's) or TranscriptImages. Partial sequences of cDNA clone inserts, so-called “expressedsequence tags” (EST's), were derived from cDNA libraries made fromprostate tumor tissues, prostate non-tumor tissues and numerous othertissues (both tumorous and non-tumorous) and entered into a database(LIFESEQ™ database, available from Incyte Pharmaceuticals, Palo Alto,Calif.) as gene transcript images. See International Publication No. WO95/20681. (A transcript image is a listing of the number of EST's foreach of the represented genes in a given tissue library. EST's sharingregions of mutual sequence overlap are classified into clusters. Acluster is assigned a clone number from a representative 5′ EST. Often,a cluster of interest can be extended by comparing its consensussequence with sequences of other EST's which did not meet the criteriafor automated clustering. The alignment of all available clusters andsingle EST's represent a contig from which a consensus sequence isderived.) The transcript images then were evaluated to identify ESTsequences that were representative primarily of the prostate tissuelibraries. These target clones then were ranked according to theirabundance (occurrence) in the target libraries and their absence frombackground libraries. Higher abundance clones with low backgroundoccurrence were given higher study priority. EST's corresponding to theconsensus sequence of PS108 were found in 82.3% (28 of 34) of prostatetissue libraries. EST's corresponding to the consensus sequence SEQUENCEID NO 16 (or fragments thereof) were found in 3.4% (21 of 618) of theother, non-prostate, libraries of the data base. Therefore, theconsensus sequence or fragment thereof was found more than 24 times moreoften in prostate than non-prostate tissues. Overlapping clones 1864683(SEQUENCE ID NO 1), 1711346 (SEQUENCE ID NO 2), 1913982 (SEQUENCE ID NO3), 825779 (SEQUENCE ID NO 4), 2626650 (SEQUENCE ID NO 5), 1808389(SEQUENCE ID NO 6), 1651121 (SEQUENCE ID NO 7), 3520833 (SEQUENCE ID NO8), 2188949 (SEQUENCE ID NO 9), 3497504 (SEQUENCE ID NO 10), 3964174(SEQUENCE ID NO 11), 3705332 (SEQUENCE ID NO 12), 2270646 (SEQUENCE IDNO 13), and 1810610 (SEQUENCE ID NO 14) were identified for furtherstudy. These represented the minimum number of clones that were neededto form the contig and from which, along with the full-length sequenceof clone 1711346IH (SEQUENCE ID NO 15), the consensus sequence providedherein (SEQUENCE ID NO 16) was derived.

B. Generation of a Consensus Sequence. The nucleotide sequences ofclones 1864683 (SEQUENCE ID NO 1), 1711346 (SEQUENCE ID NO 2), 1913982(SEQUENCE ID NO 3), 825779 (SEQUENCE ID NO 4), 2626650 (SEQUENCE ID NO5), 1808389 (SEQUENCE ID NO 6), 1651121 (SEQUENCE ID NO 7), 3520833(SEQUENCE ID NO 8), 2188949 (SEQUENCE ID NO 9), 3497504 (SEQUENCE ID NO10), 3964174 (SEQUENCE ID NO 11), 3705332 (SEQUENCE ID NO 12), 2270646(SEQUENCE ID NO 13), 1810610 (SEQUENCE ID NO 14), and 1711346IH(SEQUENCE ID NO 15), were entered in the Sequencher™ Program (availablefrom Gene Codes Corporation, Ann Arbor, Mich.) in order to generate anucleotide alignment (contig map) and then generate their consensussequence (SEQUENCE ID NO 16). FIGS. 1A-1E show the nuclcotide sequencealignment of these clones and their resultant nucleotide consensussequence (SEQUENCE ID NO 16). FIG. 2 presents the contig map depictingthe clones 1864683 (SEQUENCE ID NO 1), 1711346 (SEQUENCE ID NO 2),1913982 (SEQUENCE ID NO 3), 825779 (SEQUENCE ID NO 4), 2626650 (SEQUENCEID NO 5), 1808389 (SEQUENCE ID NO 6),1651121 (SEQUENCE ID NO 7), 3520833(SEQUENCE ID NO 8), 2188949 (SEQUENCE ID NO 9), 3497504 (SEQUENCE ID NO10), 3964174 (SEQUENCE ID NO 11), 3705332 (SEQUENCE ID NO 12), 2270646(SEQUENCE ID NO 13), 1810610 (SEQUENCE ID NO 14) and 1711346IH (SEQUENCEID NO 15) which form overlapping regions of the PS108 gene, and theresultant consensus nucleotide sequence (SEQUENCE ID NO 16) of theseclones in a graphic display. Following this, a three-frame translationwas performed on the consensus sequence (SEQUENCE ID NO 16). The firstforward frame was found to have an open reading frame encoding a 255residue amino acid sequence which is presented as SEQUENCE ID NO 36.

Analysis of the LIFESEQ™ database indicates a possible C/T polymorphismat position 823 in the consensus nucleotide sequence (SEQUENCE ID NO16). There were ten occurrences of the C nucleotide variant and threeoccurrences of the T nucleotide variant in the database. The site of thepotential polymorphism is in the 3′ untranslated region of the gene anddoes not affect the amino acid composition of the protein product.

Example 2 Sequencing of PS108 EST-Specific Clones

The full-length DNA sequence of clone 1711346IH of the PS108 gene contigwas determined (SEQUENCE ID NO 15) using dideoxy termination sequencingwith dye terminators following known methods (F. Sanger et al., PNASU.S.A. 74:5463 (1977).

Because the pINCY vector (available from Incyte Pharmaceuticals, Inc.,Palo Alto, Calif.) contains universal priming sites just adjacent to the3′ and 5′ ligation junctions of the inserts, approximately 300 bases ofthe insert were sequenced in both directions using two universal primers(SEQUENCE ID NO 19 and SEQUENCE ID NO 20, available from New EnglandBiolabs, Beverly, Mass., and Applied Biosystems Inc, Foster City,Calif., respectively). The sequencing reactions were run on apolyacrylamide denaturing gel, and the sequences were determined by anApplied Biosystems 377 Sequencer (available from Applied Biosystems,Foster City, Calif.). Additional sequencing primers (SEQUENCE ID NOS21-33) were designed from sequence information of the consensus sequence(SEQUENCE ID NO 16). These primers then were used to determine theremaining DNA sequence of the cloned insert from each DNA strand, aspreviously described.

Example 3 Nucleic Acid

A. RNA Extraction from Tissue. Total RNA was isolated from prostatetissues and from non-prostate tissues. Various methods were utilized,including, but not limited to, the lithium chloride/urea technique,known in the art and described by Kato et al. (J. Virol. 61:2182-2191,1987), and TRIzol™ (Gibco-BRL, Grand Island, N.Y.).

Briefly, tissue was placed in a sterile conical tube on ice, and 10-15volumes of 3 M LiCl, 6 M urea, 5 mM EDTA, 0.1 M β-mercaptoethanol, 50 mMTris-HCl (pH 7.5) were added. The tissue was homogenized with aPolytron® homogenizer (Brinkman Instruments, Inc., Westbury, N.Y.) for30-50 sec on ice. The solution was transferred to a 15 ml plasticcentrifuge tube and placed overnight at −20° C. The tube was centrifugedfor 90 min at 9,000×g at 0-4° C. and the supernatant was immediatelydecanted. Ten ml of 3 M LiCl were added and the tube was vortexed for 5sec. The tube was centrifuged for 45 min at 11,000×g at 0-4° C. Thedecanting, resuspension in LiCl, and centrifugation was repeated and thefinal pellet was air dried and suspended in 2 ml of 1 mM EDTA, 0.5% SDS,10 mM Tris (pH 7.5). Twenty microliters (20 μl) of Proteinase K (20mg/ml) were added, and the solution was incubated for 30 min at 37° C.with occasional mixing. One-tenth volume (0.22-0.25 ml) of 3 M NaCl wasadded, and the solution was vortexed before transfer into another tubecontaining 2 ml of phenol/chloroform/isoamyl alcohol (PCI). The tube wasvortexed for 1-3 sec and centrifuged for 20 min at 3,000×g at 10° C. ThePCI extraction was repeated and followed by two similar extractions withchloroform/isoamyl alcohol (CI). The final aqueous solution wastransferred to a prechilled 15 ml Corex glass tube containing 6 ml ofabsolute ethanol, the tube was covered with parafilm, and placed at −20°C. overnight. The tube was centrifuged for 30 min at 10,000×g at 0-4° C.and the ethanol supernatant was decanted immediately. The RNA pellet waswashed four times with 10 ml of 75% ice-cold ethanol and the finalpellet was air dried for 15 min at room temperature. The RNA wassuspended in 0.5 ml of 10 mM TE (pH 7.6, 1 mM EDTA) and itsconcentration was determined spectrophotometrically. RNA samples werealiquoted and stored at −70° C. as ethanol precipitates.

The quality of the RNA was determined by agarose gel electrophoresis(see Example 5, Northern Blot Analysis) and staining with 0.5 μg/mlethidium bromide for one hour. RNA samples that did not contain intactrRNAs were excluded from the study.

Alternatively, for RT-PCR analysis, 1 ml of Ultraspec RNA reagent wasadded to 120 mg of pulverized tissue in a 2.0 ml polypropylene microfugetube, homogenized with a Polytron® homogenizer (Brinkman Instruments,Inc., Westbury, N.Y.) for 50 sec and placed on ice for 5 min. Then, 0.2ml of chloroform was added to each sample, followed by vortexing for 15sec. The sample was placed on ice for another 5 min, followed bycentrifugation at 12,000×g for 15 min at 4° C. The upper layer wascollected and transferred to another RNase-free 2.0 ml microfuge tube.An equal volume of isopropanol was added to each sample, and thesolution was placed on ice for 10 min. The sample was centrifuged at12,000×g for 10 min at 4° C., and the supernatant was discarded. Theremaining pellet was washed twice with cold 75% ethanol, resuspended byvortexing, and the resuspended material was then pelleted bycentrifugation at 7500×g for 5 min at 4° C. Finally, the RNA pellet wasdried in a Speedvac (Savant, Farmingdale, N.Y.) for 5 min andreconstituted in RNase-free water.

B. RNA Extraction from Blood Mononuclear Cells. Mononuclear cells areisolated from blood samples from patients by centrifugation usingFicoll-Hypaque as follows. A 10 ml volume of whole blood is mixed withan equal volume of RPMI Medium (Gibco-BRL, Grand Island, N.Y.). Thismixture is then underlayed with 10 ml of Ficoll-Hypaque (Pharmacia,Piscataway, N.J.) and centrifuged for 30 minutes at 200×g. The buffycoat containing the mononuclear cells is removed, diluted to 50 ml withDulbecco's PBS (Gibco-BRL, Grand Island, N.Y.) and the mixturecentrifuged for 10 minutes at 200×g. After two washes, the resultingpellet is resuspended in Dulbecco's PBS to a final volume of 1 ml.

RNA is prepared from the isolated mononuclear cells as described by N.Kato et al., J. Virology 61: 2182-2191 (1987). Briefly, the pelletedmononuclear cells are brought to a final volume of 1 ml and then areresuspended in 250 μL of PBS and mixed with 2.5 ml of 3 M LiCl, 6 Murea, 5 mM EDTA, 0.1 M 2-mercaptoethanol, 50 mM Tris-HCl (pH 7.5). Theresulting mixture is homogenized and incubated at −20° C. overnight. Thehomogenate is centrifuged at 8,000 RPM in a Beckman J2-21M rotor for 90minutes at 0-4° C. The pellet is resuspended in 10 ml of 3 M LiCl byvortexing and then centrifuged at 10,000 RPM in a Beckman J2-21 M rotorcentrifuge for 45 minutes at 0-4° C. The resuspending and pelletingsteps are then repeated. The pellet is resuspended in 2 ml of I mM EDTA,0.5% SDS, 10 mM Tris (pH 7.5) and 400 μg Proteinase K with vortexing andthen it is incubated at 37° C. for 30 minutes with shaking. One tenthvolume of 3 M NaCl then is added and the mixture is vortexed. Proteinsare removed by two cycles of extraction with phenol/chloroform/isoamylalcohol (PCI) followed by one extraction with chloroform/isoamyl alcohol(CI). RNA is precipitated by the addition of 6 ml of absolute ethanolfollowed by overnight incubation at −20° C. After the precipitated RNAis collected by centrifugation, the pellet is washed 4 times in 75%ethanol. The pelleted RNA is then dissolved in solution containing 1 mMEDTA, 10 mM Tris-HCl (pH 7.5).

Non-GI tract tissues are used as negative controls. The mRNA can befurther purified from total RNA by using commercially available kitssuch as oligo dT cellulose spin columns (RediCol™ from Pharmacia,Uppsala, Sweden) for the isolation of poly-adenylated RNA. Total RNA ormRNA can be dissolved in lysis buffer (5 M guanidine thiocyanate, 0.1 MEDTA, pH 7.0) for analysis in the ribonuclease protection assay.

C. RNA Extraction from polysomes. Tissue is minced in saline at 4° C.and mixed with 2.5 volumes of 0.8 M sucrose in a TK₁₅₀M (150 mM KCl, 5mM MgCl₂, 50 mM Tris-HCl, pH 7.4) solution containing 6 mM2-mercaptoethanol. The tissue is homogenized in a Teflon-glass Potterhomogenizer with five strokes at 100-200 rpm followed by six strokes ina Dounce homogenizer, as described by B. Mechler, Methods in Enzymology152:241-248 (1987). The homogenate then is centrifuged at 12,000×g for15 min at 4° C. to sediment the nuclei. The polysomes are isolated bymixing 2 ml of the supernatant with 6 ml of 2.5 M sucrose in TK₁₅₀M andlayering this mixture over 4 ml of 2.5 M sucrose in TK₁₅₀M in a 38 mlpolyallomer tube. Two additional sucrose TK₁₅₀M solutions aresuccessively layered onto the extract fraction; a first layer of 13 ml2.05 M sucrose followed by a second layer of 6 ml of 1.3 M sucrose. Thepolysomes are isolated by centrifuging the gradient at 90,000×g for 5 hrat 4° C. The fraction then is taken from the 1.3 M sucrose/2.05 Msucrose interface with a siliconized pasteur pipette and diluted in anequal volume of TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA). An equal volumeof 90° C. SDS buffer (1% SDS, 200 mM NaCl, 20 mM Tris-HCl, pH 7.4) isadded and the solution is incubated in a boiling water bath for 2 min.Proteins next are digested with a Proteinase-K digestion (50 mg/ml) for15 min at 37° C. The mRNA is purified with 3 equal volumes ofphenol-chloroform extractions followed by precipitation with 0.1 volumeof 2 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol at −20° C.overnight. The precipitated RNA is recovered by centrifugation at12,000×g for 10 min at 4° C. The RNA is dried and resuspended in TE (pH7.4) or distilled water. The resuspended RNA then can be used in a slotblot or dot blot hybridization assay to check for the presence of PS108mRNA (see Example 6).

The quality of nucleic acid and proteins is dependent on the method ofpreparation used. Each sample may require a different preparationtechnique to maximize isolation efficiency of the target molecule. Thesepreparation techniques are within the skill of the ordinary artisan.

Example 4 Ribonuclease Protection Assay

A. Synthesis of Labeled Complementary RNA (cRNA) Hybridization Probe andUnlabeled Sense Strand. Labeled antisense and unlabeled sense riboprobesare transcribed from the PS108 gene cDNA sequence which contains a 5′RNA polymerase promoter such as SP6 or 17. The sequence may be from avector containing the appropriate PS108 cDNA insert, or from aPCR-generated product of the insert using PCR primers which incorporatea 5′ RNA polymerase promoter sequence. For example, the describedplasmid (clone 1281865), or another comparable clone containing thePS108 gene cDNA sequence flanked by opposed SP6 and T7 polymerasepromoters, is purified using Qiagen Plasmid Purification Kit (Qiagen,Chatsworth, Calif.). Then 10 μg of the plasmid are linearized by cuttingwith 10 U DdeI restriction enzyme for 1 hr at 37° C. The linearizedplasmid is purified using QIAprep kits (Qiagen, Chatsworth, Calif.) andused for the synthesis of antisense transcript from the appropriate SP6or T7 promoter using the Riboprobe® in vitro Transcription System(Promega Corporation, Madison, Wis.), as described by the supplier'sinstructions, incorporating either 6.3 μM (alpha³²P) CTP (Amersham LifeSciences, Inc. Arlington Heights, Ill.) or 100-500 μM biotinylated CTPas a label. To generate the sense strand, 10 μg of the purified plasmidare cut with restriction enzymes 10U XbaI and 10 U NotI, and transcribedas above from the appropriate SP6 or T7 promoter. Both sense andantisense strands are isolated by spin column chromatography. Unlabeledsense strand is quantitated by UV absorption at 260 nm.

B. Hybridization of Labeled Probe to Target. Frozen tissue is pulverizedto powder under liquid nitrogen and 100-500 mg are dissolved in 1 ml oflysis buffer, available as a component of the Direct Protect™ LysateRNase Protection kit (Ambion, Inc., Austin, Tex.). Further dissolutioncan be achieved using a tissue homogenizer. In addition, a dilutionseries of a known amount of sense strand in mouse liver lysate is madefor use as a positive control. Finally, 45 μl of solubilized tissue ordiluted sense strand is mixed directly with either: (1) 1×10⁵ cpm ofradioactively labeled probe; or (2) 250 μg of non-isotopically labeledprobe in 5 μl of lysis buffer. Hybridization is allowed to proceedovernight at 37° C. See, T. Kaabache et al., Anal. Biochem. 232:225-230(1995).

C. RNase Digestion. RNA that is not hybridized to probe is removed fromthe reaction as per the Direct Protect™ protocol using a solution ofRNase A and RNase T1 for 30 min at 37° C., followed by removal of RNaseby Proteinase-K digestion in the presence of sodium sarcosyl. Hybridizedfragments protected from digestion are then precipitated by the additionof an equal volume of isopropanol and placed at −70° C. for 3 hr. Theprecipitates are collected by centrifugation at 12,000×g for 20 min.

D. Fragment Analysis. The precipitates are dissolved in denaturing gelloading dye (80% formamide, 10 mM EDTA (pH 8.0), 1 mg/ml xylene cyanol,1 mg/ml bromophenol blue), heat denatured, and electrophoresed in 6%polyacrylamide TBE, 8 M urea denaturing gels. The gels are imaged andanalyzed using the STORM™ storage phosphor autoradiography system(Molecular Dynamics, Sunnyvale, Calif.). Quantitation of protectedfragment bands, expressed in femtograms (fg), is achieved by comparingthe peak areas obtained from the test samples to those from the knowndilutions of the positive control sense strand (see Section B, supra).The results are expressed in molecules of PS108 RNA/cell and as a imagerating score. In cases where non-isotopic labels are used, hybrids aretransferred from the gels to membranes (nylon or nitrocellulose) byblotting and then analyzed using detection systems that employstreptavidin alkaline phosphatase conjugates and chemiluminesence orchemifluoresence reagents.

Detection of a product comprising a sequence selected from the groupconsisting of SEQUENCE ID NOS 1-16 and fragments or complements thereof,is indicative of the presence of PS108 mRNAs, suggesting a diagnosis ofa prostate tissue disease or condition, such as prostate cancer.

Example 5 Northern Blotting

The northern blot technique is used to identify a specific size RNAfragment from a complex population of RNA using gel electrophoresis andnucleic acid hybridization. Northern blotting is a technique well-knownin the art. Briefly, 5-10 μg of total RNA (see Example 3) are incubatedin 15 μl of a solution containing 40 mM morphilinopropanesulfonic acid(MOPS) (pH 7.0), 10 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde,50% v/v formamide for 15 min at 65° C. The denatured RNA is mixed with 2μl of loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromophenol blue,0.4% xylene cyanol) and loaded into a denaturing 1.0% agarose gelcontaining 40 mM MOPS (pH 7.0), 10 mM sodium acetate, 1 mM EDTA and 2.2M formaldehyde. The gel is electrophoresed at 60 V for 1.5 h and rinsedin RNAse free water. RNA is transferred from the gel onto nylonmembranes (Brightstar-Plus, Ambion, Inc., Austin, Tex.) for 1.5 hoursusing the downward alkaline capillary transfer method (Chomczynski,Anal. Biochem. 201:134-139, 1992). The filter is rinsed with 1×SSC, andRNA is crosslinked to the filter using a Stratalinker (Stratagene, Inc.,La Jolla, Calif.) on the autocrosslinking mode and dried for 15 min. Themembrane is then placed into a hybridization tube containing 20 ml ofpreheated prehybridization solution (5×SSC, 50% formamide, 5×Denhardt'ssolution, 100 μg/ml denatured salmon sperm DNA) and incubated in a 42°C. hybridization oven for at least 3 hr. While the blot isprehybridizing, a ³²P-labeled random-primed probe is generated using thePS108 insert fragment (obtained by digesting clone 1711346 or anothercomparable clone with XbaI and NotI) using Random Primer DNA LabelingSystem (Life Technologies, Inc., Gaithersburg, Md.) according to themanufacturer's instructions. Half of the probe is boiled for 10 min,quick chilled on ice and added to the hybridization tube. Hybridizationis carried out at 42° C. for at least 12 hr. The hybridization solutionis discarded and the filter is washed in 30 ml of 3×SSC, 0.1% SDS at 42°C. for 15 min, followed by 30 ml of 3×SSC, 0.1% SDS at 42° C. for 15min. The filter is wrapped in Saran Wrap and exposed to Kodak XAR-Omatfilm for 8-96 hr and the film is developed for analysis.

Detection of a product comprising a sequence selected from the groupconsisting of SEQUENCE ID NOS 1-16 and fragments or complements thereof,is indicative of the presence of PS108 mRNAs, suggesting a diagnosis ofa prostate tissue disease or condition, such as prostate cancer.

Example 6 Dot Blot/Slot Blot

Dot and slot blot assays are quick methods to evaluate the presence of aspecific nucleic acid sequence in a complex mix of nucleic acid. Toperform such assays, up to 50 μg of RNA are mixed in 50 μl of 50%formamide, 7% formaldehyde, 1×SSC, incubated 15 min at 68° C., and thencooled on ice. Then, 100 μl of 20×SSC are added to the RNA mixture andloaded under vacuum onto a manifold apparatus that has a preparednitrocellulose or nylon membrane. The membrane is soaked in water,20×SSC for 1 hour, placed on two sheets of 20×SSC prewet Whatman #3filter paper, and loaded into a slot blot or dot blot vacuum manifoldapparatus. The slot blot is analyzed with probes prepared and labeled asdescribed in Example 4, supra. Detection of mRNA corresponding to asequence selected from the group consisting of SEQUENCE ID NOS 1-16 andfragments or complements thereof, is indicative of the presence of PS108mRNAs, suggesting a diagnosis of a prostate tissue disease or condition,such as prostate cancer.

Other methods and buffers which can be utilized in the methods describedin Examples 5 and 6, but not specifically detailed herein, are known inthe art and are described in J. Sambrook et al, supra which isincorporated herein by reference.

Example 7 In Situ Hybridization

This method is useful to directly detect specific target nucleic acidsequences in cells using detectable nucleic acid hybridization probes.

Tissues are prepared with cross-linking fixative agents such asparaformaldehyde or glutaraldehyde for maximum cellular RNA retention.See, L. Angerer et al., Methods in Cell Biol. 35:37-71 (1991). Briefly,the tissue is placed in greater than 5 volumes of 1% glutaraldehyde in50 mM sodium phosphate, pH 7.5 at 4° C. for 30 min. The solution ischanged with fresh glutaraldehyde solution (1% glutaraldehyde in 50 mMsodium phosphate, pH 7.5) for a further 30 min fixing. The fixingsolution should have an osmolality of approximately 0.375% NaCl. Thetissue is washed once in isotonic NaCl to remove the phosphate.

The fixed tissues then are embedded in paraffin as follows. The tissueis dehydrated though a series of increasing ethanol concentrations for15 mill each: 50% (twice), 70% (twice), 85%, 90% and then 100% (twice).Next, the tissue is soaked in two changes of xylene for 20 min each atroom temperature. The tissue is then soaked in two changes of a 1:1mixture of xylene and paraffin for 20 min each at 60° C.; and then inthree final changes of paraffin for 15 min each.

Next, the tissue is cut in 5 μm sections using a standard microtome andplaced on a slide previously treated with a tissue adhesive such as3-aminopropyltriethoxysilane.

Paraffin is removed from the tissue by two 10 min xylene soaks andrehydrated in a series of decreasing ethanol concentrations: 99%(twice), 95%, 85%, 70%, 50%, 30%, and then in distilled water (twice).The sections are pre-treated with 0.2 M HCl for 10 min and permeabilizedwith 2 μg/ml Proteinase-K at 37° C. for 15 min.

Labeled Riboprobes transcribed from the PS108 gene plasmid (see Example4) are hybridized to the prepared tissue sections and incubatedovernight at 56° C. in 3×standard saline extract and 50% formamide.Excess probe is removed by washing in 2×standard saline citrate and 50%formamide followed by digestion with 100 μg/ml RNase A at 37° C. for 30min. Fluorescence probe is visualized by illumination with ultraviolet(UV) light under a microscope. Fluorescence in the cytoplasm isindicative of PS108 mRNA. Alternatively, the sections can be visualizedby autoradiography.

Detection of a product comprising a sequence selected from the groupconsisting of SEQUENCE ID NOS 1-16 and fragments or complements thereof,is indicative of the presence of PS108 mRNAs, suggesting a diagnosis ofa prostate tissue disease or condition, such as prostate cancer.

Example 8 Reverse Transcription PCR

A. One Step RT-PCR Assay. Target-specific primers are designed to detectthe above-described target sequences by reverse transcription PCR usingmethods known in the art. One step RT-PCR is a sequential procedure thatperforms both RT and PCR in a single reaction mixture. The procedure isperformed in a 200 μl reaction mixture containing 50 mM(N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15, 81.7 mM KOAc, 33.33 mM KOH,0.01 mg/ml bovine serum albumin, 0.1 mM ethylene diaminetetraaceticacid, 0.02 mg/ml NaN₃, 8% w/v glycerol, 150 μM each of dNTP, 0.25 μMeach primer, 5U rTth polymerase, 3.25 mM Mn(OAc)₂ and 5 μl of target RNA(see Example 3). Since RNA and the rTth polymerase enzyme are unstablein the presence of Mn(OAc)₂, the Mn(OAc)₂ should be added just beforetarget addition. Optimal conditions for cDNA synthesis and thermalcycling readily can be determined by those skilled in the art. Thereaction is incubated in a Perkin-Elmer Thermal Cycler 480. Optimalconditions for cDNA synthesis and thermal cycling can readily bedetermined by those skilled in the art. Conditions which may be founduseful include cDNA synthesis at 60°-70° C. for 15-45 min and 30-45amplification cycles at 94° C., 1 min; 55°-70° C., 1 min; 72° C., 2 min.One step RT-PCR also may be performed by using a dual enzyme procedurewith Taq polymerase and a reverse transcriptase enzyme, such as MMLV orAMV RT enzymes.

B. Traditional RT-PCR. A traditional two-step RT-PCR reaction wasperformed, as described by K. Q. Hu et al., Virology 181:721-726 (1991).Briefly, 1.0 μg of extracted mRNA (see Example 3) was reversetranscribed in a 20 μl reaction mixture containing 1×PCR II buffer(Perkin-Elmer), 5 mM MgCl₂, 1 mM dNTP, 20 U RNasin, 2.5 μM randomhexamers, and 50 U MMLV (Moloney murine leukemia virus) reversetranscriptase (RT). Reverse transcription was performed at roomtemperature for 10 min, 42° C. for 30 min in a PE-480 thermal cycler,followed by further incubation at 95° C. for 5 min to inactivate the RT.PCR was performed using 2 μl of the cDNA reaction in a final PCRreaction volume of 50 μl containing 10 mM Tris-HCl (pH 8.3),50 mM KCl,1.5 mM MgCl₂, 200 μM dNTP, 0.4 μM of each sense and antisense primer(SEQUENCE ID NO 34 and SEQUENCE ID NO 35, respectively), and 2.5 U ofTaq polymerase. The reaction was incubated in an MJ Research ModelPTC-200 as follows: Denaturation at 94° C. for 2 min. followed by 35cycles of amplification (94° C., 45 sec; 62 ° C., 45 sec; 72° C., 2 min); a final extension (72° C., 5 min); and a soak at 4° C.

C. PCR Fragment Analysis. The correct products were verified by sizedetermination using gel electrophoresis with a SYBR® Green I nucleicacid gel stain (Molecular Probes, Eugene, Oreg.). Gels were stained withSYBR® Green I at a 1:10,000 dilution in 1×TBE for 30 minutes. Gels wereimaged using a STORM imaging system (FIGS. 3A-3B). More particularly,FIG. 3A shows a 344 bp PS108-specific PCR amplification product in lanes3-6 and lanes 9-10. The 344 hp PS108-specific amplicon was present in 2of 4 prostate cancer tissue RNAs (lanes 5 and 9) and in 3 of 3 BPHprostate tissue RNAs (lanes 3, 4, and 6). (The BPH sample in lane 10 wasa diluted duplicate of the sample in lane 6.) The human placental DNAcontrol (lane 2) was negative in the analysis, suggesting that theamplicons in lanes 3-6 and lanes 9-10 were the result of amplificationof mRNA and not DNA. As shown in FIG. 3B, the 344 bp amplicon wasdetected in RNAs from BPH and prostate cancer tissues (lanes 3 and 4,respectively) and in one of two colon cancer tissues (lane 5). ThisRNA-specific product was not observed in human placental DNA (lane 2).Further, this RNA-specific product was not observed in RNAs isolatedfrom a different colon cancer tissue sample (lane 6), a normal colontissue sample (lane 7), breast cancer tissue samples (lanes 8-9), anormal breast tissue sample (lane 10), normal lung tissue samples (lane11-12) or a lung cancer tissue sample (lane 13).

Detection of a product comprising a sequence selected from the groupconsisting of SEQUENCE ID NOS 1-16 and fragments or complements thereof,is indicative of the presence of PS108 mRNAs, suggesting a diagnosis ofa prostate tissue disease or condition, such as prostate cancer.

Example 9 OH-PCR

A. Probe selection and Labeling. Target-specific primers and probes aredesigned to detect the above-described target sequences byoligonucleotide hybridization PCR. International Publication Nos WO92/10505, published Jun. 25, 1992, and WO 92/11388, published Jul. 9,1992, teach methods for labeling oligonucleotides at their 5′ and 3′ends, respectively. According to one known method for labeling anoligonucleotide, a label-phosphoramidite reagent is prepared and used toadd the label to the oligonucleotide during its synthesis. For example,see N. T. Thuong et al., Tet. Letters 29(46):5905-5908 (1988); or J. S.Cohen et al., published U.S. patent application Ser. No. 07/246,688(NTIS ORDER No. PAT-APPL-7-246,688) (1989). Preferably, probes arelabeled at their 3′ end to prevent participation in PCR and theformation of undesired extension products. For one step OH-PCR, theprobe should have a T_(M) at least 15° C. below the T_(M) of theprimers. The primers and probes are utilized as specific bindingmembers, with or without detectable labels, using standardphosphoramidite chemistry and/or post-synthetic labeling methods whichare well-known to one skilled in the art.

B. One Step Oligo Hybridization PCR. OH-PCR is performed on a 200 μlreaction containing 50 mM (N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15,81.7 mM KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1 mMethylene diaminetetraacetic acid, 0.02 mg/ml NaN₃, 8% w/v glycerol, 150μM each of dNTP, 0.25 μM each primer, 3.75 nM probe, 5U rTth polymerase,3.25 mM Mn(OAc)₂ and 5 μl blood equivalents of target (see Example 3).Since RNA and the rTth polymerase enzyme are unstable in the presence ofMn(OAc)₂, the Mn(OAc)₂ should be added just before target addition. Thereaction is incubated in a Perkin-Elmer Thermal Cycler 480. Optimalconditions for cDNA synthesis and thermal cycling can be readilydetermined by those skilled in the art. Conditions which may be founduseful include cDNA synthesis (60° C., 30 min), 30-45 amplificationcycles (94° C., 40 sec; 55-70° C., 60 sec), oligo-hybridization (97° C.,5 min; 15° C., 5 min; 15° C. soak). The correct reaction productcontains at least one of the strands of the PCR product and aninternally hybridized probe.

C. OH-PCR Product Analysis. Amplified reaction products are detected onan LCx® analyzer system (available from Abbott Laboratories, AbbottPark, Ill.). Briefly, the correct reaction product is captured by anantibody labeled microparticle at a capturable site on either the PCRproduct strand or the hybridization probe, and the complex is detectedby binding of a detectable antibody conjugate to either a detectablesite on the probe or the PCR strand. Only a complex containing a PCRstrand hybridized with the internal probe is detectable. The detectionof this complex then is indicative of the presence of PS108 mRNA,suggesting a diagnosis of a prostate disease or condition, such asprostate cancer.

Many other detection formats exist which can be used and/or modified bythose skilled in the art to detect the presence of amplified ornon-amplified PS108-derived nucleic acid sequences including, but notlimited to, ligase chain reaction (LCR, Abbott Laboratories, AbbottPark, Ill.); Q-beta replicase (Gene-Trak™, Naperville, Ill.), branchedchain reaction (Chiron, Emeryville, Calif.) and strand displacementassays (Becton Dickinson, Research Triangle Park, N.C.).

Detection of a product comprising a sequence selected from the groupconsisting of SEQUENCE ID NOS 1-16 and fragments or complements thereof,is indicative of the presence of PS108 mRNAs, suggesting a diagnosis ofa prostate tissue disease or condition, such as prostate cancer.

Example 10 Synthetic Peptide Production

Synthetic peptides were modeled and then prepared based upon thepredicted amino acid sequence of the PS108 polypeptide consensussequence (see Example 1). In particular, a number of PS108 peptidesderived from SEQUENCE ID NO 36 were prepared, including the peptides ofSEQUENCE ID NO 37, SEQUENCE ID NO 38, and SEQUENCE ID NO 39. Allpeptides were synthesized on a Symphony Peptide Synthesizer (availablefrom Rainin Instrument Co, Emeryville, Calif.) using FMOC chemistry,standard cycles and in-situ HBTU activation. Cleavage and deprotectionconditions were as follows: a volume of 2.5 ml of cleavage reagent(77.5% v/v trifluoroacetic acid, 15% v/v ethanedithiol, 2.5% v/v water,5% v/v thioanisole, 1-2% w/v phenol) was added to the resin, andagitated at room temperature for 2-4 hours. Then the filtrate wasremoved and the peptide was precipitated from the cleavage reagent withcold diethyl ether. Each peptide was filtered, purified viareverse-phase preparative HPLC using a water/acetonitrile/0.1% TFAgradient, and lyophilized. The product was confirmed by massspectrometry.

The purified peptides were used to immunize animals (see Example 14).

Example 11a Expression of Protein in a Cell Line Using Plasmid 577

A. Construction of a PS108 Expression Plasmid. Plasmid 577, described inU.S. patent application Ser. No. 08/478,073, filed Jun. 7, 1995 andincorporated herein by reference, has been constructed for theexpression of secreted antigens in a permanent cell line. This plasmidcontains the following DNA segments: (a) a 2.3 Kb fragment of pBR322containing bacterial beta-lactamase and origin of DNA replication; (b) a1.8 Kb cassette directing expression of a neomycin resistance gene undercontrol of HSV-1 thymidine kinase promoter and poly-A addition signals;(c) a 1.9 Kb cassette directing expression of a dihydrofolate reductasegene under the control of an SV-40 promoter and poly-A addition signals;(d) a 3.5 Kb cassette directing expression of a rabbit immunoglobulinheavy chain signal sequence fused to a modified hepatitis C virus (HCV)E2 protein under the control of the Simian Virus 40 T-Ag promoter andtranscription enhancer, the hepatitis B virus surface antigen (HBsAg)enhancer I followed by a fragment of Herpes Simplex Virus-1 (HSV-1)genome providing poly-A addition signals; and (e) a residual 0.7 Kbfragment of Simian Virus 40 genome late region of no function in thisplasmid. All of the segments of the vector were assembled by standardmethods known to those skilled in the art of molecular biology.

Plasmids for the expression of secretable PS108 proteins are constructedby replacing the hepatitis C virus E2 protein coding sequence in plasmid577 with that of a PS108 polynucleotide sequence selected from the groupconsisting of SEQUENCE ID NOS 1-16 and fragments or complements thereof,as follows. Digestion of plasmid 577 with XbaI releases the hepatitis Cvirus E2 gene fragment. The resulting plasmid backbone allows insertionof the PS108 cDNA insert downstream of the rabbit immunoglobulin heavychain signal sequence which directs the expressed proteins into thesecretory pathway of the cell. The PS108 cDNA fragment is generated byPCR using standard procedures. Encoded in the sense PCR primer sequenceis an XbaI site, immediately followed by a 12 nucleotide sequence thatencodes the amino acid sequence Ser-Asn-Glu-Leu (“SNEL”) to promotesignal protease processing, efficient secretion and final productstability in culture fluids. Immediately following this 12 nucleotidesequence, the primer contains nucleotides complementary to templatesequences encoding amino acids of the PS108 gene. The antisense primerincorporates a sequence encoding the following eight amino acids justbefore the stop codons: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQUENCE ID NO40). Within this sequence is incorporated a recognition site to aid inanalysis and purification of the PS108 protein product. A recognitionsite (termed “FLAG”) that is recognized by a commercially availablemonoclonal antibody designated anti-FLAG M2 (Eastman Kodak, Co., NewHaven, Conn.) can be utilized, as well as other comparable sequences andtheir corresponding antibodies. For example, PCR is performed usingGeneAmp® reagents obtained from Perkin-Elmer-Cetus, as directed by thesupplier's instructions. PCR primers are used at a final concentrationof 0.5 μM. PCR is performed on the PS108 plasmid template in a 100 μlreaction for 35 cycles (94° C., 30 seconds; 55° C., 30 seconds; 72° C.,90 seconds) followed by an extension cycle of 72° C. for 10 min.

B. Transfection of Dihydrofolate Reductase Deficient Chinese HamsterOvary Cells. The plasmid described supra is transfected intoCHO/dhfr-cells (DXB-111, Uriacio et al., PNAS 77:4451-4466 (1980)).These cells are available from the A.T.C.C., 12301 Parklawn Drive,Rockville, Md. 20852, under Accession No. CRL 9096. Transfection iscarried out using the cationic liposome-mediated procedure described byP. L. Feigner et al., PNAS 84:7413-7417 (1987). Particularly,CHO/dhfr-cells are cultured in Ham's F-12 media supplemented with 10%fetal calf serum, L-glutamine (1 mM) and freshly seeded into a flask ata density of 5-8×10⁵ cells per flask. The cells are grown to aconfluency of between 60 and 80% for transfection. Twenty micrograms (20μg) of plasmid DNA is added to 1.5 ml of Opti-MEM I medium and 100 μl ofLipofectin Reagent (Gibco-BRL; Grand Island, N.Y.) are added to a second1.5 ml portion of Opti-MEM I media. The two solutions are mixed andincubated at room temperature for 20 min. After the culture medium isremoved from the cells, the cells are rinsed 3 times with 5 ml ofOpti-MEM I medium. The Opti-MEM I-Lipofection-plasmid DNA solution thenis overlaid onto the cells. The cells are incubated for 3 h at 37° C.,after which time the Opti-MEM I-Lipofectin-DNA solution is replaced withculture medium for an additional 24 h prior to selection.

C. Selection and Amplification. One day after transfection, cells arepassaged 1:3 and incubated with dhfr/G418 selection medium (hereafter,“F-12 minus medium G”). Selection medium is Ham's F-12 with L-glutamineand without hypoxanthine, thymidine and glycine (JRH Biosciences,Lenexa, Kans.) and 300 μg ml G418 (Gibco-BRL; Grand Island, N.Y.). Mediavolume-to-surface area ratios of 5 ml per 25 cm² are maintained. Afterapproximately two weeks, DHFR/G418 cells are expanded to allow passageand continuous maintenance in F-12 minus medium G.

Amplification of each of the transfected PS108 cDNA sequences isachieved by stepwise selection of DHFR⁺, G418⁺ cells with methotrexate[reviewed by R. Schimke, Cell 37:705-713 (1984)]. Cells are incubatedwith F-12 minus medium G containing 150 nM methotrexate (MTX) (Sigma,St. Louis, Mo.) for approximately two weeks until resistant coloniesappear. Further gene amplification is achieved by selection of 150 nMadapted cells with 5 μM MTX.

D. Antigen Production. F-12 minus medium G supplemented with 5 μM MTX isoverlaid onto just confluent monolayers for 12 to 24 h at 37° C. in 5%CO₂. The growth medium is removed and the cells are rinsed 3 times withDulbecco's phosphate buffered saline (PBS) (with calcium and magnesium)(Gibco-BRL; Grand Island, N.Y.) to remove the remaining media/serumwhich may be present. Cells then are incubated with VAS custom medium(VAS custom formulation with L-glutamine with HEPES without phenol red,available from JRH Bioscience; Lenexa, Kans., product number 52-08678P),for 1 h at 37° C. in 5% CO₂. Cells then are overlaid with VAS forproduction at 5 ml per T flask. Medium is removed after seven days ofincubation, retained, and then frozen to await purification withharvests 2, 3 and 4. The monolayers are overlaid with VAS for 3 moreseven day harvests.

E. Analysis of Prostate Tissue Gene PS108 Antigen Expression. Aliquotsof VAS supernatants from the cells expressing the PS108 proteinconstruct are analyzed, either by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) using standard methods and reagents known in the art (Laemmlidiscontinuous gels), or by mass spectrometry.

F. Purification. Purification of the PS108 protein containing the FLAGsequence is performed by immunoaffinity chromatography using an affinitymatrix comprising anti-FLAG M2 monoclonal antibody covalently attachedto agarose by hydrazide linkage (Eastman Kodak Co., New Haven, Conn.).Prior to affinity purification, protein in pooled VAS medium harvestsfrom roller bottles is exchanged into 50 mM Tris-HCl (pH 7.5), 150 mMNaCl buffer using a Sephadex G-25 (Pharmacia Biotech Inc., Uppsala,Sweden) column. Protein in this buffer is applied to the anti-FLAG M2antibody affinity column. Non-binding protein is eluted by washing thecolumn with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl buffer. Bound proteinis eluted using an excess of FLAG peptide in 50 mM Tris-HCl (pH 7.5),150 mM NaCl. The excess FLAG peptide can be removed from the purifiedPS108 protein by gel electrophoresis or HPLC.

Although plasmid 577 is utilized in this example, it is known to thoseskilled in the art that other comparable expression systems, such asCMV, can be utilized herein with appropriate modifications in reagentand/or techniques and are within the skill of the ordinary artisan.

The largest cloned insert containing the coding region of the PS108 geneis then sub-cloned into either (i) a eukaryotic expression vector whichmay contain, for example, a cytomegalovirus (CMV) promoter and/orprotein fusible sequences which aid in protein expression and detection,or (ii) a bacterial expression vector containing a superoxide-dismutase(SOD) and CMP-KDO synthetase (CKS) or other protein fusion gene forexpression of the protein sequence. Methods and vectors which are usefulfor the production of polypeptides which contain fusion sequences of SODare described in EPO 0196056, published Oct. 1, 1986, which isincorporated herein by reference and those containing fusion sequencesof CKS are described in EPO Publication No. 0331961, published Sep. 13,1989, which publication is also incorporated herein by reference. Thisso-purified protein can be used in a variety of techniques, including,but not limited to animal immunization studies, solid phaseimmunoassays, etc.

Example 11b Expression of Protein in a Cell Line Using pcDNA3.1/Myc-His

A. Construction of a PS108 Expression Plasmid. Plasmid pcDNA3.1/Myc-His(Cat. #V855-20, Invitrogen, Carlsbad, Calif.) was developed for theexpression of secreted antigens by most mammalian cell lines. Expressedprotein inserts are fused to a myc-his peptide tag. The myc-his tag is a21 residue amino acid sequence having the following sequence:Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-Asn-Met-His-Thr-Glu-His-His-His-His-His-His(SEQUENCE ID NO 41) and comprises a c-myc oncoprotein epitope and apolyhistidine sequence which are useful for the purification of anexpressed fusion protein by using either anti-myc or anti-his affinitycolumns, or metalloprotein binding columns.

Plasmids for the expression of secretable PS108 proteins are constructedby inserting a PS108 polynucleotide sequence selected from the groupconsisting of SEQUENCE ID NOS 1-16 and fragments or complements thereof.Prior to construction of a PS108 expression plasmid, the PS108 cDNAsequence is first cloned into a pCR®-Blunt vector as follows.

The PS108 cDNA fragment is generated by PCR using standard procedures.For example, PCR is performed using Stratagene® reagents obtained fromStratagene, as directed by the supplier's instructions. PCR primers areused at a final concentration of 0.5 μM. PCR using 5 U of pfu polymerase(Stratagene, La Jolla, Calif.) is performed on the PS108 plasmidtemplate (see Example 2) in a 50 μl reaction for 30 cycles (94° C., 1min; 65° C., 1.5 min; 72° C., 3 min) followed by an extension cycle of72° C. for 8 min. (The sense PCR primer sequence comprises nucleotideswhich are either complementary to the pINCY vector directly upstream ofthe PS108 gene insert or which incorporate a 5′ EcoRI restriction site,an adjacent downstream protein translation consensus initiator, and a 3′nucleic acid sequence which is the same sense as the 5′-most end of thePS108 cDNA insert. The antisense primer incorporates a 5′ NotIrestriction sequence and a sequence complementary to the 3′ end of thePS108 cDNA insert just upstream of the 3′-most, in-frame stop codon.)Five microliters (5 μl) of the resulting blunted-ended PCR product areligated into 25 ng of linearized pCR®-Blunt vector (Invitrogen,Carlsbad, Calif.) interrupting the lethal ccdB gene of the vector. Theresulting ligated vector is transformed into TOP10 E. coli (Invitrogen,Carlsbad, Calif.) using a One Shot™ transformation kit (Invitrogen,Carlsbad, Calif.) following supplier's directions. The transformed cellsare grown on LB-Kan (50 μg/ml kanamycin) selection plates at 37° C. Onlycells containing a plasmid with an interrupted ccdB gene will grow aftertransformation (Grant, S. G. N., PNAS 87:4645-4649 (1990)). Transformedcolonies are picked and grown up in 3 ml of LB-Kan broth at 37° C.Plasmid DNA is isolated by using a QIAprep® (Qiagen Inc., Santa Clarita,Calif.) procedure, as directed by the supplier's instructions. The DNAis cut with EcoRI or SnaBI, and NotI restriction enzymes to release thePS108 insert fragment. The fragment is run on 1% Seakem® LE agarose/0.5μg/ml ethidium bromide/TE gel, visualized by UV irradiation, excised andpurified using QIAquick™ (Qiagen Inc., Santa Clarita, Calif.)procedures, as directed by the supplier's instructions.

The pcDNA3.1/Myc-His plasmid DNA is linearized by digestion with EcoRIor SnaBI, and NotI in the polylinker region of the plasmid DNA. Theresulting plasmid DNA backbone allows insertion of the PS108 purifiedcDNA fragment, supra, downstream of a CMV promoter which directsexpression of the proteins in mammalian cells. The ligated plasmid istransformed into DH5 alpha™ cells (GibcoBRL Gaithersburg, Md.), asdirected by the supplier's instructions. Briefly, 10 ng ofpcDNA3.1/Myc-His containing a PS108 insert are added to 50 μl ofcompetent DH5 alpha cells, and the contents are mixed gently. Themixture is incubated on ice for 30 min, heat shocked for 20 sec at 37°C., and placed on ice for an additional 2 min. Upon addition of 0.95 mlof LB medium, the mixture is incubated for 1 h at 37° C. while shakingat 225 rpm. The transformed cells then are plated onto 100 mm LB/Amp (50μg/ml ampicillin) plates and grown at 37° C. Colonies are picked andgrown in 3 ml of LB/Amp broth. Plasmid DNA is purified using a QIAprepkit. The presence of the insert is confirmed using techniques known tothose skilled in the art, including, but not limited to restrictiondigestion and gel analysis. (J. Sambrook et al., supra.)

B. Transfection of Human Embryonic Kidney Cell 293 Cells. The PS108expression plasmid described in section A, supra, is retransformed intoDH5 alpha cells, plated onto LB/ampicillin agar, and grown up in 10 mlof LB/ampicillin broth, as described hereinabove. The plasmid ispurified using a QIAfilter™ Maxi kit (Qiagen, Chatsworth, Calif.) and istransfected into HEK293 cells (F. L. Graham et al., J. Gen. Vir.36:59-72 (1977)). These cells are available from the A.T.C.C., 12301Parklawn Drive, Rockville, Md. 20852, under Accession No. CRL 1573.Transfection is carried out using the cationic lipofectamine-mediatedprocedure described by P. Hawley-Nelson et al., Focus 15.73 (1993).Particularly, HEK293 cells are cultured in 10 ml DMEM media supplementedwith 10% fetal bovine serum (FBS), L-glutamine (2 mM) and freshly seededinto 100 mm culture plates at a density of 9×10⁶ cells per plate. Thecells are grown at 37 ° C. to a confluency of between 70% and 80% fortransfection. Eight micrograms (8 μg) of plasmid DNA are added to 800 μlof Opti-MEM I® medium (Gibco-BRL, Grand Island, N.Y.), and 48-96 μl ofLipofectamine™ Reagent (Gibco-BRL, Grand Island, N.Y.) are added to asecond 800 μl portion of Opti-MEM I media. The two solutions are mixedand incubated at room temperature for 15-30 min. After the culturemedium is removed from the cells, the cells are washed once with 10 mlof serum-free DMEM. The Opti-MEM I-Lipofectamine-plasmid DNA solution isdiluted with 6.4 ml of serum-free DMEM and then overlaid onto the cells.The cells are incubated for 5 h at 37° C., after which time, anadditional 8 ml of DMEM with 20% FBS are added. After 18-24 h, the oldmedium is aspirated, and the cells are overlaid with 5 ml of fresh DMEMwith 5% FBS. Supernatants and cell extracts are analyzed for PS108 geneactivity 72 h after transfection.

C. Analysis of Prostate Tissue Gene PS108 Antigen Expression. Theculture supernatant, supra, is transferred to cryotubes and stored onice. HEK293 cells are harvested by washing twice with 10 ml of coldDulbecco's PBS and lysing by addition of 1.5 ml of CAT lysis buffer(Boehringer Mannheim, Indianapolis, Ind.), followed by incubation for 30min at room temperature. Lysate is transferred to 1.7 ml polypropylenemicrofuge tubes and centrifuged at 1000×g for 10 min. The supernatant istransferred to new cryotubes and stored on ice. Aliquots of supernatantsfrom the cells and the lysate of the cells expressing the PS108 proteinconstruct are analyzed for the presence of recombinant PS108 protein.The aliquots can be run on SDS-polyacrylamide gel electrophoresis(SDS-PAGE) using standard methods and reagents known in the art. (J.Sambrook et al., supra). These gels can then be blotted onto a solidmedium such as nitrocellulose, nytran, etc., and the PS108 protein bandcan be visualized using western blotting techniques with anti-mycepitope or anti-histidine monoclonal antibodies (Invitrogen, Carlsbad,Calif.) or anti-PS108 polyclonal serum (see Example 14). Alternatively,the expressed PS108 recombinant protein can be analyzed by massspectrometry (see Example 12).

D. Purification. Purification of the PS108 recombinant proteincontaining the myc-his sequence is performed using the Xpress® affinitychromatography system (Invitrogen, Carlsbad, Calif.) containing anickel-charged agarose resin which specifically binds polyhistidineresidues. Supernatants from 10×100 mm plates, prepared as describedsupra, are pooled and passed over the nickel-charged column. Non-bindingprotein is eluted by washing the column with 50 mM Tris-HCl (pH 7.5)/150mM NaCl buffer, leaving only the myc-his fusion proteins. Bound PS108recombinant protein then is eluted from the column using either anexcess of imidazole or histidine, or a low pH buffer. Alternatively, therecombinant protein can also be purified by binding at the myc-hissequence to an affinity column consisting of either anti-myc oranti-histidine monoclonal antibodies conjugated through a hydrazide orother linkage to an agarose resin and eluting with an excess of mycpeptide or histidine, respectively.

The purified recombinant protein can then be covalently cross-linked toa solid phase, such as N-hydroxysuccinimide-activated sepharose columns(Pharmacia Biotech, Piscataway, N.J.), as directed by supplier'sinstructions. These columns containing covalently linked PS108recombinant protein, can then be used to purify anti-PS108 antibodiesfrom rabbit or mouse sera (see Examples 13 and 14).

E. Coating Microtiter Plates with PS108 Expressed Proteins. Supernatantfrom a 100 mm plate, as described supra, is diluted 1:3 in PBS. Then,100 μl of the resulting mixture are placed into each well of aReacti-Bind™ metal chelate microtiter plate (Pierce, Rockford, Ill.),incubated at room temperature while shaking, and is followed by fourwashes with deionized water. The prepared microtiter plate can then beused to screen polyclonal antisera for the presence of PS108 antibodies(see Example 17).

Although pcDNA3.1/Myc-His is utilized in this example, it is known tothose skilled in the art that other comparable expression systems can beutilized herein with appropriate modifications in reagent and/ortechniques and are within the skill of one of ordinary skill in the art.The largest cloned insert containing the coding region of the PS108 geneis sub-cloned into either (i) a eukaryotic expression vector which maycontain, for example, a cytomegalovirus (CMV) promoter and/or proteinfusible sequences which aid in protein expression and detection, or (ii)a bacterial expression vector containing a superoxide-dismutase (SOD)and CMP-KDO synthetase (CKS) or other protein fusion gene for expressionof the protein sequence. Methods and vectors which are useful for theproduction of polypeptides which contain fusion sequences of SOD aredescribed in published EPO application No. EP 0 196 056, published Oct.1, 1986, which is incorporated herein by reference, and vectorscontaining fusion sequences of CKS are described in published EPOapplication No. EP 0 331 961, published Sep. 13, 1989, which publicationis also incorporated herein by reference. The purified protein can beused in a variety of techniques, including, but not limited to animalimmunization studies, solid phase immunoassays, etc.

Example 12 Chemical Analysis of Prostate Tissue Proteins

A. Analysis of Tryptic Peptide Fragments Using MS. Sera from patientswith prostate disease, such as prostate cancer, sera from patients withno prostate disease, extracts of prostate tissues or cells from patientswith prostate disease, such as prostate cancer, extracts of prostatetissues or cells from patients with no prostate disease, and extracts oftissues or cells from other non-diseased or diseased organs of patients,are run on a polyacrylamide gel using standard procedures and stainedwith Coomassie Blue. Sections of the gel suspected of containing theunknown polypeptide are excised and subjected to an in-gel reduction,acetamidation and tryptic digestion. P. Jeno et al, Anal. Bio.224:451-455 (1995) and J. Rosenfeld et al, Anal. Bio. 203:173-179(1992). The gel sections are washed with 100 mM NH₄HCO₃ andacetonitrile. The shrunken gel pieces are swollen in digestion buffer(50 mM NH₄HCO₃, 5 mM CaCl₂ and 12.5 μg/ml trypsin) at 4° C. for 45 min.The supernatant is aspirated and replaced with 5 to 10 μl of digestionbuffer without trypsin and allowed to incubate overnight at 37° C.Peptides are extracted with 3 changes of 5% formic acid and acetonitrileand evaporated to dryness. The peptides are adsorbed to approximately0.1 μl of POROS R2 sorbent (Perseptive Biosystems, Framingham, Mass.)trapped in the tip of a drawn gas chromatography capillary tube bydissolving them in 10 μl of 5% formic acid and passing it through thecapillary. The adsorbed peptides are washed with water and eluted with5% formic acid in 60% methanol. The eluant is passed directly into thespraying capillary of an API III mass spectrometer (Perkin-Elmer Sciex,Thornhill, Ontario, Canada) for analysis by nano-electrospray massspectrometry. M. Wilm et al., Int. J. Mass Spectrom. Ion Process136:167-180 (1994) and M. Wilm et al., Anal. Chem. 66:1-8 (1994). Themasses of the tryptic peptides are determined from the mass spectrumobtained off the first quadrupole. Masses corresponding to predictedpeptides can be further analyzed in MS/MS mode to give the amino acidsequence of the peptide.

B. Peptide Fragment Analysis Using LC/MS. The presence of polypeptidespredicted from mRNA sequences found in hyperplastic disease tissues alsocan be confirmed using liquid chromatography/tandem mass spectrometry(LC/MS/MS). D. Hess et al., METHODS. A Companion to Methods inEnzymology 6:227-238 (1994). The serum specimen or tumor extract fromthe patient is denatured with SDS and reduced with dithiothreitol (1.5mg/ml) for 30 min at 90° C. followed by alkylation with iodoacetamide (4mg/ml) for 15 min at 25° C. Following acrylamide electrophoresis, thepolypeptides are electroblotted to a cationic membrane and stained withCoomassie Blue. Following staining, the membranes are washed andsections thought to contain the unknown polypeptides are cut out anddissected into small pieces. The membranes are placed in 500 μlmicrocentrifuge tubes and immersed in 10 to 20 μl of proteolyticdigestion buffer (100 mM Tris-HCl, pH 8.2, containing 0.1 M NaCl, 10%acetonitrile, 2 mM CaCl₂ and 5 μg/ml trypsin) (Sigma, St. Louis, Mo.).After 15 h at 37° C., 3 μl of saturated urea and 1 μl of 100 μg/mltrypsin are added and incubated for an additional 5 h at 37° C. Thedigestion mixture is acidified with 3 μl of 10% trifluoroacetic acid andcentrifuged to separate supernatant from membrane. The supernatant isinjected directly onto a microbore, reverse phase HPLC column and elutedwith a linear gradient of acetonitrile in 0.05% trifluoroacetic acid.The eluate is fed directly into an electrospray mass spectrometer, afterpassing though a stream splitter if necessary to adjust the volume ofmaterial. The data is analyzed following the procedures set forth inExample 12, Section A.

Example 13 Gene Immunization Protocol

A. In Vivo Antigen Expression. Gene immunization circumvents proteinpurification steps by directly expressing an antigen in vivo afterinoculation of the appropriate expression vector. Also, production ofantigen by this method may allow correct protein folding andglycosylation since the protein is produced in mammalian tissue. Themethod utilizes insertion of the gene sequence into a plasmid whichcontains a CMV promoter, expansion and purification of the plasmid andinjection of the plasmid DNA into the muscle tissue of an animal.Preferred animals include mice and rabbits. See, for example, H. Daviset al., Human Molecular Genetics 2:1847-1851 (1993). After one or twobooster immunizations, the animal can then be bled, ascites fluidcollected, or the animal's spleen can be harvested for production ofhybridomas.

B. Plasmid Preparation and Purification. PS108 cDNA sequences aregenerated from the PS108 cDNA-containing vector using appropriate PCRprimers containing suitable 5′ restriction sites following theprocedures described in Example 11. The PCR product is cut withappropriate restriction enzymes and inserted into a vector whichcontains the CMV promoter (for example, pRc/CMV or pcDNA3 vectors fromInvitrogen, San Diego, Calif.). This plasmid then is expanded in theappropriate bacterial strain and purified from the cell lysate using aCsCl gradient or a Qiagen plasmid DNA purification column. All thesetechniques are familiar to one of ordinary skill in the art of molecularbiology.

C. Immunization Protocol. Anesthetized animals are immunizedintramuscularly with 0.1-100 μg of the purified plasmid diluted in PBSor other DNA uptake enhancers (Cardiotoxin, 25% sucrose). See, forexample, H. Davis et al, Human Gene Therapy 4:733-740 (1993); and P. W.Wolff et al, Biotechniques 11:474-485 (1991). One to two boosterinjections are given at monthly intervals.

D. Testing and Use of Antiserum. Animals are bled and the resultant seratested for antibody using peptides synthesized from the known genesequence (see Example 16) using techniques known in the art, such aswestern blotting or EIA techniques. Antisera produced by this method canthen be used to detect the presence of the antigen in a patient's tissueor cell extract or in a patient's serum by ELISA or Western blottingtechniques, such as those described in Examples 15 through 18.

Example 14 Production of Antibodies Against PS108

A. Production of Polyclonal Antisera. Antiserum against PS108 wasprepared by injecting rabbits with peptides whose sequences were derivedfrom that of the predicted amino acid sequence (SEQUENCE ID NO 36) ofthe PS108 consensus nucleotide sequence. The synthesis of PS108 peptides(SEQUENCE ID NO 37, SEQUENCE ID NO 38, and SEQUENCE ID NO 39) isdescribed in Example 10. Peptides used as immunogens were not conjugatedto a carrier such as keyhole limpet hemocyanine, KLH, (i.e., they wereunconjugated.).

Animal Immunization. Female white New Zealand rabbits weighing 2 kg ormore were used for raising polyclonal antiserum. One animal wasimmunized per unconjugated peptide (SEQUENCE ID NO 37, SEQUENCE ID NO38, and SEQUENCE ID NO 39). One week prior to the first immunization, 5to 10 ml blood samples were obtained from each animal to serve as anon-immune prebleed sample.

The unconjugated PS108 peptides of SEQUENCE ID NO 37, SEQUENCE ID NO 38,and SEQUENCE ID NO 39, were used to prepare the primary immunogen byemulsifying 0.5 ml of the peptide at a concentration of 2 mg/ml in PBS(pH 7.2) which contained 0.5 ml of complete Freund's adjuvant (CFA)(Difco, Detroit, Mich.). The immunogen was injected into several sitesof the animal via subcutaneous, intraperitoneal, and intramuscularroutes of administration. Four weeks following the primary immunization,a booster immunization was administered. The immunogen used for thebooster immunization dose was prepared by emulsifying 0.5 ml of the sameunconjugated peptides used for the primary immunogen, except that thepeptide now was diluted to 1 mg/ml with 0.5 ml of incomplete Freund'sadjuvant (IFA) (Difco, Detroit, Mich.). Again, the booster dose wasadministered into several sites via subcutaneous, intraperitoneal andintramuscular types of injections. The animals were bled (5 ml) twoweeks after the booster immunizations and each serum was tested forimmunoreactivity to the peptide as described below. The booster andbleed schedules were repeated at 4 week intervals until an adequatetiter was obtained. The titer or concentration of antiserum wasdetermined by using unconjugated peptides in a microtiter EIA asdescribed in Example 17, below. An antibody titer of 1:500 or greaterwas considered an adequate titer for further use and study.

TABLE 1 Titer of rabbit anti-PS108 peptide antisera (11 week bleed)Peptide Immunogen Titer SEQUENCE ID NO 37 250,000  SEQUENCE ID NO 3856,000 SEQUENCE ID NO 39 53,000

B. Production of Monoclonal Antibody.

1. Immunization Protocol. Mice are immunized using peptides which caneither be conjugated to a carrier such as KLH [prepared as describedhereinbelow, or unconjugated (i.e., not conjugated to a carrier such asKLH)], except that the amount of the unconjugated or conjugated peptidefor monoclonal antibody production in mice is one-tenth the amount usedto produce polyclonal antisera in rabbits. Thus, the primary immunogenconsists of 100 μg of unconjugated or conjugated peptide in 0.1 ml ofCFA emulsion while the immunogen used for booster immunizations consistsof 50 μg of unconjugated or conjugated peptide in 0.1 ml of IFA.Hybridomas for the generation of monoclonal antibodies are prepared andscreened using standard techniques. The methods used for monoclonalantibody development follow procedures known in the art such as thosedetailed in Kohler and Milstein, Nature 256:494 (1975) and reviewed inJ. G. R. Hurrel, ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla. (1982). Another methodof monoclonal antibody development which is based on the Kohler andMilstein method is that of L. T. Mimms et al., Virology 176:604-619(1990), which is incorporated herein by reference.

The immunization regimen (per mouse) consists of a primary immunizationwith additional booster immunizations. The primary immunogen used forthe primary immunization consists of 100 μg of unconjugated orconjugated peptide in 50 μl of PBS (pH 7.2) previously emulsified in 50μl of CFA. Booster immunizations performed at approximately two weeks-and four weeks-post primary immunization consist of 50 μg ofunconjugated or conjugated peptide in 50 μl of PBS (pH 7.2) emulsifiedwith 50 μl IFA. A total of 100 μl of this immunogen are inoculatedintraperitoneally and subcutaneously into each mouse. Individual miceare screened for immune response by microtiter plate enzyme immunoassay(EIA) as described in Example 17 approximately four weeks after thethird immunization. Mice are inoculated either intravenously,intrasplenically or intraperitoneally with 50 μg of unconjugated orconjugated peptide in PBS (pH 7.2) approximately fifteen weeks after thethird immunization.

Three days after this intravenous boost, splenocytes are fused with, forexample, Sp2/0-Ag14 myeloma cells (Milstein Laboratories, England) usingthe polyethylene glycol (PEG) method. The fusions are cultured inIscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal calfserum (FCS), plus 1% hypoxanthine, aminopterin and thymidine (HAT). Bulkcultures are screened by microtiter plate EIA following the protocol inExample 17. Clones reactive with the peptide used as an immunogen andnon-reactive with other peptides (i.e., peptides of PS108 not used asthe immunogen) are selected for final expansion. Clones thus selectedare expanded, aliquoted and frozen in IMDM containing 10% FCS and 10%dimethyl sulfoxide, (DMSO).

2. Peptide Conjugation. Peptide is conjugated to maleimide activated KLH(commercially available as Imject®, available from Pierce ChemicalCompany, Rockford, Ill.). Imject® contains about 250 moles of reactivemaleimide groups per mole of hemocyanine. The activated KLH is dissolvedin phosphate buffered saline (PBS, pH 8.4) at a concentration of about7.7 mg/mi. The peptide is conjugated through cysteines occurring in thepeptide sequence, or to a cysteine previously added, to the synthesizedpeptide in order to provide a point of attachment. The peptide isdissolved in DMSO (Sigma Chemical Company, St. Louis, Mo.) and reactedwith the activated KLH at a mole ratio of about 1.5 moles of peptide permole of reactive maleimide attached to the KLH. A procedure for theconjugation of peptide is provided hereinbelow. It is known to theordinary artisan that the amounts, times and conditions of such aprocedure can be varied to optimize peptide conjugation.

The conjugation reaction described hereinbelow is based on obtaining 3mg of KLH peptide conjugate (“conjugated peptide”), which contains about0.77 μmoles of reactive maleimide groups. This quantity of peptideconjugate usually is adequate for one primary injection and four boosterinjections for production of polyclonal antisera in a rabbit. Briefly,peptide is dissolved in DMSO at a concentration of 1.16 μmoles/100 μl ofDMSO. One hundred microliters (100 μl) of the DMSO solution are added to380 μl of the activated KLH solution prepared as described hereinabove,and 20 μl of PBS (pH 8.4) are added to bring the volume to 500 μl. Thereaction is incubated overnight at room temperature with stirring. Theextent of reaction is determined by measuring the amount of unreactedthiol in the reaction mixture. The difference between the startingconcentration of thiol and the final concentration is assumed to be theconcentration of peptide which has coupled to the activated KLH. Theamount of remaining thiol is measured using Ellman's reagent(5,5′-dithiobis(2-nitrobenzoic acid), Pierce Chemical Company, Rockford,Ill.). Cysteine standards are made at a concentration of 0, 0.1, 0.5, 2,5 and 20 mM by dissolving 35 mg of cysteine HCl (Pierce ChemicalCompany, Rockford, Ill.) in 10 ml of PBS (pH 7.2) and diluting the stocksolution to the desired concentrations. The photometric determination ofthe concentration of thiol is accomplished by placing 200 μl of PBS (pH8.4) in each well of an Immulon 2® microwell plate (Dynex Technologies,Chantilly, Va.). Next, 10 μl of standard or reaction mixture are addedto each well. Finally, 20 μl of Ellman's reagent at a concentration of 1mg/ml in PBS (pH 8.4) are added to each well. The wells are incubatedfor 10 minutes at room temperature, and the absorbance of all wells isread at 415 nm with a microplate reader (such as the BioRad Model 3550,BioRad, Richmond, Calif.). The absorbance of the standards is used toconstruct a standard curve and the thiol concentration of the reactionmixture is determined from the standard curve. A decrease in theconcentration of free thiol is indicative of a successful conjugationreaction. Unreacted peptide is removed by dialysis against PBS (pH 7.2)at room temperature for 6 hours. The conjugate is stored at 2-8° C. ifit is to be used immediately; otherwise, it is stored at −20° C. orcolder.

3. Production of Ascites Fluid Containing Monoclonal Antibodies. Frozenhybridoma cells prepared as described hereinabove are thawed and placedinto expansion culture. Viable hybridoma cells are inoculatedintraperitoneally into Pristane treated mice. Ascitic fluid is removedfrom the mice, pooled, filtered through a 0.2μ filter and subjected toan immunoglobulin class G (IgG) analysis to determine the volume of theProtein A column required for the purification.

4. Purification of Monoclonal Antibodies From Ascites Fluid. Briefly,filtered and thawed ascites fluid is mixed with an equal volume ofProtein A sepharose binding buffer (1.5 M glycine, 3.0 M NaCl, pH 8.9)and refiltered through a 0.2μ filter. The volume of the Protein A columnis determined by the quantity of IgG present in the ascites fluid. Theeluate then is dialyzed against PBS (pH 7.2) overnight at 2-8° C. Thedialyzed monoclonal antibody is sterile filtered and dispensed inaliquots. The immunoreactivity of the purified monoclonal antibody isconfirmed by determining its ability to specifically bind to the peptideused as the immunogen by use of the EIA microtiter plate assay procedureof Example 17. The specificity of the purified monoclonal antibody isconfirmed by determining its lack of binding to irrelevant peptides suchas peptides of PS108 not used as the immunogen. The purified anti-PS108monoclonal thus prepared and characterized is placed at either 2-8° C.for short term storage or at −80° C. for long term storage.

5. Further Characterization of Monoclonal Antibody. The isotype andsubtype of the monoclonal antibody produced as described hereinabove canbe determined using commercially available kits (available fromAmersham. Inc., Arlington Heights, Ill.). Stability testing also can beperformed on the monoclonal antibody by placing an aliquot of themonoclonal antibody in continuous storage at 2-8° C. and assayingoptical density (OD) readings throughout the course of a given period oftime.

C. Use of Recombinant Proteins as immunogens. It is within the scope ofthe present invention that recombinant proteins made as described hereincan be utilized as immunogens in the production of polyclonal andmonoclonal antibodies, with corresponding changes in reagents andtechniques known to those skilled in the art.

Example 15 Purification of Serum Antibodies Which Specifically Bind toPS108 Peptides

Immune sera, obtained as described hereinabove in Examples 13 and/or 14,is affinity purified using immobilized synthetic peptides prepared asdescribed in Example 10, or recombinant proteins prepared as describedin Example 11. An IgG fraction of the antiserum is obtained by passingthe diluted, crude antiserum over a Protein A column (Affi-Gel proteinA, Bio-Rad, Hercules, Calif.). Elution with a buffer (Binding Buffer,supplied by the manufacturer) removes substantially all proteins thatare not immunoglobulins. Elution with 0.1 M buffered glycine (pH 3)gives an immunoglobulin preparation that is substantially free ofalbumin and other serum proteins.

Immunoaffinity chromatography is performed to obtain a preparation witha higher fraction of specific antigen-binding antibody. The peptide usedto raise the antiserum is immobilized on a chromatography resin, and thespecific antibodies directed against its epitopes are adsorbed to theresin. After washing away non-binding components, the specificantibodies are eluted with 0.1 M glycine buffer, pH 2.3. Antibodyfractions are immediately neutralized with 1.0 M Tris buffer (pH 8.0) topreserve immunoreactivity. The chromatography resin chosen depends onthe reactive groups present in the peptide. If the peptide has an aminogroup, a resin such as Affi-Gel 10 or Affi-Gel 15 is used (Bio-Rad,Hercules, Calif.). If coupling through a carboxy group on the peptide isdesired, Affi-Gel 102 can be used (Bio-Rad, Hercules, Calif.). If thepeptide has a free sulfhydryl group, an organomercurial resin such asAffi-Gel 501 can be used (Bio-Rad, Hercules, Calif.).

Alternatively, spleens can be harvested and used in the production ofhybridomas to produce monoclonal antibodies following routine methodsknown in the art as described hereinabove.

Example 16 Western Blotting of Tissue Samples

Protein extracts were prepared by homogenizing tissue samples in 0.1 MTris-HCl (pH 7.5), 15% (w/v) glycerol, 0.2 mM EDTA, 1.0 mM1,4-dithiothreitol, 10 μg/ml leupeptin and 1.0 mMphenylmethylsulfonylfluoride (S. R. Kain et al., Biotechniques 17:982(1994). Following homogenization, the homogenates were centrifuged at 4°C. for 5 minutes to separate supernatant from debris. For proteinquantitation, 3-10 μl of supernatant were added to 1.5 ml ofbicinchoninic acid reagent (Sigma, St. Louis, Mo.), and the resultingabsorbance at 562 nm were measured.

For SDS-PAGE, samples were adjusted to desired protein concentrationwith Tricine Buffer (Novex, San Diego, Calif.), mixed with an equalvolume of 2×Tricine sample buffer (Novex, San Diego, Calif.), and heatedfor 5 minutes at 100° C. in a thermal cycler. Samples were then appliedto a Novex 10-20% Precast Tricine Gel for electrophoresis. Followingelectrophoresis, samples were transferred from the gels tonitrocellulose membranes in Novex Tris-Glycine Transfer buffer.Membranes were then probed with specific anti-peptide antibodies usingthe reagents and procedures provided in the Western Lights Plus orWestern Lights (Tropix, Bedford, Mass.) chemiluminescence detectionkits. Chemiluminescent bands were visualized by exposing the developedmembranes to Hyperfilm ECL (Amersham, Arlington Heights, Ill.).

FIG. 4 shows the results of the Western blot performed on a panel oftissue extracts using antiserum prepared against the PS108 peptide ofSEQUENCE ID NO 37 (see Example 14). Each lane of FIG. 4 represents adifferent tissue protein extract: (1) colon cancer; (2-4) BPH; (5)prostate cancer; (6 and 7) lung; (8 and 9) breast cancer; and (10)markers. A band above 200 kD (identified by the arrow in FIG. 4), asdetermined by protein size markers (lane 10), was detected in theprostate cancer tissue extract (lane 5) and one of the three BPH tissueextracts (lane 4).

Competition experiments were performed in an analogous manner as abovewith the following exception: the primary antibodies (anti-peptidepolyclonal antisera) were pre-incubated overnight at 4° C. with varyingconcentrations of peptide immunogen prior to exposure to thenitrocellulose filter. Development of the Western was continued asabove. Antibody binding to the band above 200 kD was inhibited at aconcentration of 2.09 μM of the synthetic peptide (SEQUENCE ID NO 37).

After visualization of the bands on film, the bands were also visualizeddirectly on the membranes by the addition and development of chromogenicsubstrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP). This chromogenicsolution contained 0.016% BCIP in a solution containing 100 mM NaCl, 5mM MgCl₂ and 100 mM Tris-HCl, pH 9.5. The filter was incubated in thesolution at room temperature until the bands developed to the desiredintensity. An image was generated by transillumination of the blot on anAlpha Innotech Corporation (San Leandro, Calif.) lightbox and capturedwith an Alpha Imager 2000 Documentation and Analysis system. Molecularmass determination was made based upon the mobility of pre-stainedmolecular weight standards (Novex, San Diego, Calif.) and biotinylatedmolecular weight standards (Tropix, Bedford, Mass.).

Example 17 EIA Microtiter Plate Assay

The immunoreactivity of antiserum obtained from rabbits as described inExample 14 was determined by means of a microtiter plate EIA, asfollows. Briefly, synthetic peptides, SEQUENCE ID NO 37, SEQUENCE ID NO38, and SEQUENCE ID NO 39, prepared as described in Example 10, weredissolved in carbonate buffer (50 mM, pH 9.6) to a final concentrationof 2 μg/ml. Next, 100 μl of the peptide or protein solution were placedin each well of an Immulon 2® microtiter plate (Dynex Technologies,Chantilly, Va.). The plate was incubated overnight at room temperatureand then washed four times with deionized water. The wells were blockedby adding 125 μl of a suitable protein blocking agent, such asSuperblock® (Pierce Chemical Company, Rockford, Ill.), to each well andthen immediately discarding the solution. This blocking procedure wasperformed three times. Antiserum obtained from immunized rabbits ormice, prepared as previously described, was diluted in a proteinblocking agent (e.g., a 3% Superblock® solution) in PBS containing 0.05%Tween-20® (monolaurate polyoxyethylene ether) (Sigma Chemical Company,St. Louis, Mo.) and 0.05% sodium azide at dilutions of 1:100, 1:500,1:2500, 1:12,500, and 1:62,500 and placed in each well of the coatedmicrotiter plate. The wells then were incubated for three hours at roomtemperature. Each well was washed four times with deionized water. Onehundred microliters of alkaline phosphatase-conjugated goat anti-rabbitIgG or goat anti-mouse IgG antiserum (Southern Biotech, Birmingham,Ala.) diluted 1:2000 in 3% Superblock® solution in phosphate bufferedsaline containing 0.05% Tween 20® and 0.05% sodium azide, were added toeach well. The wells were incubated for two hours at room temperature.Next, each well was washed four times with deionized water. One hundredmicroliters of paranitrophenyl phosphate substrate (Kirkegaard and PerryLaboratories, Gaithersburg, Md.) then were added to each well. The wellswere incubated for thirty minutes at room temperature. The absorbance at405 nm was read in each well. Positive reactions were identified by anincrease in absorbance at 405 nm in the test well above that absorbancegiven by a non-immune serum (negative control). A positive reaction wasindicative of the presence of detectable anti-PS108 antibodies. Titersof the anti-peptide antisera were calculated from the previouslydescribed dilutions of antisera and defined as the calculated dilution,where A_(405nm)=0.5 OD.

Example 18 Coating of Solid Phase Particles

A. Coating of Microparticles with Antibodies Which Specifically Bind toPS108 Antigen. Affinity purified antibodies which specifically bind toPS108 protein (see Example 15) are coated onto microparticles ofpolystyrene, carboxylated polystyrene, polymethylacrylate or similarparticles having a radius in the range of about 0.1 to 20 μm.Microparticles may be either passively or actively coated. One coatingmethod comprises coating EDAC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (AldrichChemical Co., Milwaukee, Wis.) activated carboxylated latexmicroparticles with antibodies which specifically bind to PS108 protein,as follows. Briefly, a final 0.375% solid suspension of resin washedcarboxylated latex microparticles (available from Bangs Laboratories,Carmel, Ind. or Serodyn, Indianapolis, Ind.) are mixed in a solutioncontaining 50 mM MES buffer, pH 4.0 and 150 μg/ml of affinity purifiedanti-PS108 antibody (see Example 14) for 15 min in an appropriatecontainer. EDAC coupling agent is added to a final concentration of 5.5μg/ml to the mixture and mixed for 2.5 h at room temperature.

The microparticles then are washed with 8 volumes of a Tween 20®/sodiumphosphate wash buffer (pH 7.2) by tangential flow filtration using a 0.2μm Microgon Filtration module. Washed microparticles are stored in anappropriate buffer which usually contains a dilute surfactant andirrelevant protein as a blocking agent, until needed.

B. Coating of ¼ Inch Beads. Antibodies which specifically bind toPS108-antigen also may be coated on the surface of ¼ inch polystyrenebeads by routine methods known in the art (Snitman et al, U.S. Pat. No.5,273,882, incorporated herein by reference) and used in competitivebinding or EIA sandwich assays.

Polystyrene beads first are cleaned by ultrasonicating them for about 15seconds in 10 mM NaHCO₃ buffer at pH 8.0. The beads then are washed indeionized water until all fines are removed. Beads then are immersed inan antibody solution in 10 mM carbonate buffer, pH 8 to 9.5. Theantibody solution can be as dilute as 1 μg/ml in the case of highaffinity monoclonal antibodies or as concentrated as about 500 μg/ml forpolyclonal antibodies which have not been affinity purified. Beads arecoated for at least 12 hours at room temperature, and then they arewashed with deionized water. Beads may be air dried or stored wet (inPBS, pH 7.4). They also may be overcoated with protein stabilizers (suchas sucrose) or protein blocking agents used as non-specific bindingblockers (such as irrelevant proteins, Carnation skim milk, Superblock®,or the like).

Example 19 Microparticle Enzyme Immunoassay (MEIA)

PS108 antigens are detected in patient test samples by performing astandard antigen competition EIA or antibody sandwich EIA and utilizinga solid phase such as microparticles (MEIA). The assay can be performedon an automated analyzer such as the IMx® Analyzer (Abbott Laboratories,Abbott Park, Ill.).

A. Antibody Sandwich EIA. Briefly, samples suspected of containing PS108antigen are incubated in the presence of anti-PS108 antibody-coatedmicroparticles (prepared as described in Example 17) in order to formantigen/antibody complexes. The microparticles then are washed and anindicator reagent comprising an antibody conjugated to a signalgenerating compound (i.e., enzymes such as alkaline phosphatase orhorseradish peroxide) is added to the antigen/antibody complexes or themicroparticles and incubated. The microparticles are washed and thebound antibody/antigen/antibody complexes are detected by adding asubstrate (e.g., 4-methyl umbelliferyl phosphate (MUP), or OPD/peroxide,respectively), that reacts with the signal generating compound togenerate a measurable signal. An elevated signal in the test sample,compared to the signal generated by a negative control, detects thepresence of PS108 antigen. The presence of PS108 antigen in the testsample is indicative of a diagnosis of a prostate disease or condition,such as prostate cancer.

B. Competitive Binding Assay. The competitive binding assay uses apeptide or protein that generates a measurable signal when the labeledpeptide is contacted with an anti-peptide antibody coated microparticle.This assay can be performed on the IMx® Analyzer (available from AbbottLaboratories, Abbott Park, Ill.). The labeled peptide is added to thePS108 antibody-coated microparticles (prepared as described in Example17) in the presence of a test sample suspected of containing PS108antigen, and incubated for a time and under conditions sufficient toform labeled PS108 peptide (or labeled protein)/bound antibody complexesand/or patient PS108 antigen/bound antibody complexes. The PS108 antigenin the test sample competes with the labeled PS108 peptide (or PS108protein) for binding sites on the microparticle. PS108 antigen in thetest sample results in a lowered binding of labeled peptide and antibodycoated microparticles in the assay since antigen in the test sample andthe PS108 peptide or PS108 protein compete for antibody binding sites. Alowered signal (compared to a control) indicates the presence of PS108antigen in the test sample. The presence of PS108 antigen suggests thediagnosis of a prostate disease or condition, such as prostate cancer.

The PS108 polynucleotides and the proteins encoded thereby which areprovided and discussed hereinabove are useful as markers of prostatetissue disease, especially prostate cancer. Tests based upon theappearance of this marker in a test sample such as blood, plasma orserum can provide low cost, non-invasive, diagnostic information to aidthe physician to make a diagnosis of cancer, to help select a therapyprotocol, or to monitor the success of a chosen therapy. This marker mayappear in readily accessible body fluids such as blood, urine or stoolas antigens derived from the diseased tissue which are detectable byimmunological methods. This marker may be elevated in a disease state,altered in a disease state, or be a normal protein of the prostate whichappears in an inappropriate body compartment.

Example 20 Immunohistochemical Detection of PS108 Protein

Antiserum against the PS108 peptide of SEQUENCE ID NO 37, prepared asdescribed in Example 14, supra, was used to immunohistochemically staina variety of normal and diseased tissues using standard procedures.Briefly, frozen blocks of tissue were cut into 6 micron sections, andplaced on microscope slides. After fixation in cold acetone, thesections were dried at room temperature, then washed with phosphatebuffered saline and blocked. The slides were incubated with the peptideof SEQUENCE ID NO 37 at a dilution of 1 to 500, washed, incubated withbiotinylated goat anti-rabbit antibody, washed again, and incubated withavidin labeled with horse radish peroxidase. After a final wash, theslides were incubated with 3-amino-9-ethylcarbazole substrate whichgives a red stain. The slides were counterstained with hematoxylin,mounted, and examined under a microscope by a pathologist.

Anti-PS108 antibody gives intense cytoplasmic immunoreactivity in theepithelium of all prostate specimens (benign and malignant). Secretorycells of benign glands showed strongest immunoreactivity in thesupranuclear cytoplasm. Basal cells and atrophic glands were virtuallyall negative. Cancer cells showed more extensive immunoreactivitycompared to benign epithelium. High grade prostate cancer showedextensive and intense immunoreactivity. There was almost no backgroundstaining in the stroma. For all other tissue specimens (colon, lung,liver, bladder, and breast) the immunoreactivity was weak and equivocal.

Results of all tissues examined are tabulated in Table 2. The columnlabeled “#pos/total” refers to the number of specimens positive out ofthe total number of specimens. The column labeled “%” reports thepercent of cells which are positive averaged over all tissues examined.

TABLE 2 Tissue Staining by Anti-PS108 #POS/ TISSUE TOTAL % COMMENTSColon, normal 4/4 53 Weak immunoreactivity only on superficial cells.Equivocal in one case. Colon, cancer 3/3 37 Weak immunoreactivity.Patchy in one case. Lung, normal 0/4 0 Weak immunoreactivity in 10% ofminor salivary glands in one case. Lung, bronchial 0/4 0 Noimmunoreactive cells epithelium Liver, normal 0/2 0 Intracytoplasmicbackground hepatocytes Liver, normal bile 0/2 0 duct epithelium Bladder,normal 2/3 10 Weak, equivocal immunoreactivity epithelium only onsuperficial urothelial cells. Bladder, normal wall 0/5 0 Smooth musclecells negative. Breast, benign tissue 2/2 90 Weak, cytoplasmic immuno-reactivity Breast, fibrocystic 1/1 60 Weak, equivocal epithelial changesimmunoreactivity Prostate, benign 16/16 66 Intense, granular,supranuclear cytoplasmic immunoreactivity of secretory cells. Prostate,cancer 15/15 87 Intense cytoplasmic immuno- reactivity in cancer cells.High grade cancer shows extensive and intense immunoreactivity

41 258 base pairs nucleic acid single linear not provided 1 GGGGCTGTACCAGGGCGTGC CCAGAGCTGA GCCGGGCACC GAGGCCCGGA GACACTATGA 60 TGAAGGCGTTCGGATGGGCA GCCTGGGGCT GTTCCTGCAG TGCGCCATCT CCCTGGTCTT 120 CTCTCTGGTCATGGACCGGC TGGTGCAGCG ATTCGGCACT CGAGCAGTCT ATTTGGCCAG 180 TGTGGCAGCTTTCCCTGTGG CTGCCGGTGC CACATGCCTG TCCCACAGTG TGGCCGTGGT 240 GACAGCTTCAGCCGCCTT 258 217 base pairs nucleic acid single linear not provided 2ACCAGGGCGT GCCCAGAGCT GAGCCGGGCA CCGAGGCCCG GAGACACTAT GATGAAGGCG 60TTCGGATGGG CAGCCTGGGG CTGTTCCTGC AGTGCGCCAT CTCCCTGGTC TTCTCTCTGG 120TCATGGACCG GCTGGTGCAG CGATTCGGCA CTCGAGCAGT CTATTTGGCC AGTGTGGCAG 180CTTTCCCTGT GGCTGCCGGT GCCACATGCC TGTCCCA 217 255 base pairs nucleic acidsingle linear not provided base_polymorphism 215 /note= “ N′ representsan A or G or T or C polymorphism at this position” 3 GACAGCTTCAGCCGCCCTCA CCGGGTTCAC CTTCTCAGCC CTGCAGATCC TGCCCTACAC 60 ACTGGCCTCCCTCTACCACC GGGAGAAGCA GGTGTTCCTG CCCAAATACC GAGGGGACAC 120 TGGAGGTGCTAGCAGTGAGG ACAGCCTGAT GACCAGCTTC CTGCCAGGCC CTAAGCCTGG 180 AGCTCCCTTCCCTAATGGAC ACGTGGGTGC TGGANGCAGT GGCCTGCTCC CACCTCCACC 240 CGCGCTCTGCGGGGC 255 247 base pairs nucleic acid single linear not provided 4GCTCCCTTCC CTAATGGACA CGTGGGTGCT GGAGGCAGTG GCCTGCTCCC ACCTCCACCC 60GCGCTCTGCG GGGCCTCTGC CTGTGATGTC TCCGTACGTG TGGTGGTGGG TGAGCCCACC 120GAGGCCAGGG TGGTTCCGGG CCGGGGCATC TGCCTGGACC TCGCCATCCT GGATAGTGCC 180TTCCTGCTGT CCCAGGTGGC CCCATCCCTG TTTATGGGCT CCATTGTCCA GCTCAGCCAG 240TCTGTCA 247 231 base pairs nucleic acid single linear not provided 5TGGATAGTGC CTTCCTGCTG TCCCAGGTGG CCCCATCCCT GTTTATGGGC TCCATTGTCC 60AGCTCAGCCA GTCTGTCACT GCCTATATGG TGTCTGCCGC AGGCTGGGTC TGGTCGCCAT 120TTACTTTGCT ACACAGGTAG TATTTGACAA GAGCGACTTG GCCAAATACT CAGCGTAGAA 180AACTTCCAGC ACATTGGGGT GGAGGGCCTG CCTCACTGGG TCCCAGCTCC C 231 195 basepairs nucleic acid single linear not provided base_polymorphism 178/note= “ N′ represents an A or G or T or C polymorphism at thisposition” 6 CTTGGCCAAA TACTCAGCGT AGAAAACTTC CAGCACATTG GGGTGGAGGGCCTGCCTCAC 60 TGGGTCCCAG CTCCCCGCTC CTGTTAGCCC CATGGGGCTG CCGGGCTGGCCGCCAGTTTC 120 TGTTGCTGCC AAAGTAATGT GGCTCTCTGC TGCCACCCTG TGCTGCTGAGGTGCGTANTG 180 CACAGCTGGG GGCTG 195 223 base pairs nucleic acid singlelinear not provided base_polymorphism 67 /note= “ N′ represents an A orG or T or C polymorphism at this position” base_polymorphism 222 /note=“ N′ represents an A or G or T or C polymorphism at this position” 7GCCAGTTTCT GTTGCTGCCA AAGTAATGTG GCTCTCTGCT GCCACCCTGT GCTGCTGAGG 60TGCGTANTGC ACAGCTGGGG GCTGGGGCGT CCCTCTCCTC TCTCCCCAGT CTCTAGGGCT 120GCCTGACTGG AGGCCTTCCA AGGGGGTTTC AGTCTGGACT TATACAGGGA GGCCAGAAGG 180GCTCCATGCA CTGGAATGCG GGGACTCTGC AGGTGGATTA CNC 223 342 base pairsnucleic acid single linear not provided base_polymorphism 323 /note= “N′ represents an A or G or T or C polymorphism at this position” 8GCCAGAAGGG CTCCATGCAC TGGAATGCGG GGACTCTGCA GGTGGATTAC CCAGGCTCAG 60GGTTAACAGC TAGCCTCCTA GTTGAGACAC ACCTAGAGAA GGGTTTTTGG GAGCTGAATA 120AACTCAGTCA CCTGGTTTCC CATCTCTAAG CCCCTTAACC TGCAGCTTCG TTTAATGTAG 180CTCTTGCATG GGAGTTTCTA GGATGAAACA CTCCTCCATG GGATTTGAAC ATATGAAAGT 240TATTTGTAGG GGAAGAGTCC TGAGGGGCAA CACACAAGAA CCAGGTCCCC TCAGCCCACA 300GCACTGTCTT TTTGCTGATC CANCCCCCTC TTACTTTTAT CA 342 265 base pairsnucleic acid single linear not provided 9 GGGGAAGAGT CCTGAGGGGCAACACACAAG AACCAGGTCC CCTCAGCCCA CAGCACTGTC 60 TTTTTGCTGA TCCACCCCCCTCTTACCTTT TATCAGGATG TGGCCTGTTG GTCCTTCTGT 120 TGCCATCACA GAGACACAGGCATTTAAATA TTTAACTTAT TTATTTAACA AAGTAGAAGG 180 GAATCCATTG CTAGCTTTTCTGTGTTGGTG TCTAATATTT GGGTAGGGTG GGGGATCCCC 240 AACAATCAGG TCCCCTGAGATAGCT 265 288 base pairs nucleic acid single linear not providedbase_polymorphism 147 /note= “ N′ represents an A or G or T or Cpolymorphism at this position” 10 CTCTTACCTT TTATCAGGAT GTGGCCTGTTGGTCCTTCTG TTGCCATCAC AGAGACACAG 60 GCATTTAAAT ATTTAACTTA TTTATTTAACAAAGTAGAAG GGAATCCATT GCTAGCTTTT 120 CTGTGTTGGT GTCTAATATT TGGGTANGGTGGGGGATCCC CAACAATCAG GTCCCCTGAG 180 ATAGCTGGTC ATTGGGCTGA TCATTGCCAGAATCTTCTTC TCCTGGGGTC TGGCCCCCCA 240 AAATGCCTAA CCCAGGACCT TGGAAATTCTACTCATCCCA AATGATAA 288 272 base pairs nucleic acid single linear notprovided base_polymorphism 216 /note= “ N′ represents an A or G or T orC polymorphism at this position” 11 AAATTCTACT CATCCCAAAT GATAATTCCAAATGCTGTTA CCCAAGGTTA GGGTGTTGAA 60 GGAAGGTAGA GGGTGGGGCT TCAGGTCTCAACGGCTTCCC TAACCACCCC TCTTCTCTTG 120 GCCCAGCCTG GTTCCCCCCA CTTCCACTCCCCTCTACTCT CTCTAGGACT GGGCTGATGA 180 AGGCACTGCC CAAAATTTCC CCTACCCCCAACTTTNCCCT ACCCCCAACT TTCCCCACCA 240 GCTCCACAAC CCTGTTTGGA GCTACTGCAG GT272 294 base pairs nucleic acid single linear not providedbase_polymorphism 18 /note= “ N′ represents an A or G or T or Cpolymorphism at this position” base_polymorphism 19 /note= “ N′represents an A or G or T or C polymorphism at this position”base_polymorphism 197 /note= “ N′ represents an A or G or T or Cpolymorphism at this position” 12 AAGGCACTGC CCAAAATNNC CCCTACCCCCAACTTTCCCC TACCCCCAAC TTTCCCCACC 60 AGCTCCACAA CCCTGTTTGG AGCTACTGCAGGACCAGAAG CACAAAGTGC GGTTTCCCAA 120 GCCTTTGTCC ATCTCAGCCC CCAGAGTATATCTGTGCTTG GGGAATCTCA CACAGAAACT 180 CAGGAGCACC CCCTGCNTGA GCTAAGGGAGGTCTTATCTC TCAGGGGGGG TTTAAGTGCC 240 GTTTGCAATA ATGTCGTCTT ATTTATTTAGCGGGGTGAAT ATTTTATACT GTAA 294 151 base pairs nucleic acid single linearnot provided base_polymorphism 113 /note= “ N′ represents an A or G or Tor C polymorphism at this position” base_polymorphism 147 /note= “ N′represents an A or G or T or C polymorphism at this position” 13CTCCACAACC CTGTTTGGAG CTACTGCAGG ACCAGAAGCA CAAAGTGCGG TTTCCCAAGC 60CTTTGTCCAT CTCAGCCCCC AGAGTATATC TGTGCTTGGG GAATCTCACA CANAAACTCA 120GGAGCACCCC CTGCCTGAGC TAAGGGNGGT C 151 213 base pairs nucleic acidsingle linear not provided 14 CCCAGAGTAT ATCTGTGCTT GGGGAATCTCACACAGAAAC TCAGGAGCAC CCCCTGCCTG 60 AGCTAAGGGA GGTCTTATCT CTCAGGGGGGGTTTAAGTGC CGTTTGCAAT AATGTCGTCT 120 TATTTATTTA GCGGGGTGAA TATTTTATACTGTAAGTGAG CAATCAGAGT ATAATGTTTA 180 TGGTGACAAA ATTAAAGGCT TTCTTATATGTTT 213 2143 base pairs nucleic acid single linear not provided 15ACCAGGGCGT GCCCAGAGCT GAGCCGGGCA CCGAGGCCCG GAGACACTAT GATGAAGGCG 60TTCGGATGGG CAGCCTGGGG CTGTTCCTGC AGTGCGCCAT CTCCCTGGTC TTCTCTCTGG 120TCATGGACCG GCTGGTGCAG CGATTCGGCA CTCGAGCAGT CTATTTGGCC AGTGTGGCAG 180CTTTCCCTGT GGCTGCCGGT GCCACATGCC TGTCCCACAG TGTGGCCGTG GTGACAGCTT 240CAGCCGCCCT CACCGGGTTC ACCTTCTCAG CCCTGCAGAT CCTGCCCTAC ACACTGGCCT 300CCCTCTACCA CCGGGAGAAG CAGGTGTTCC TGCCCAAATA CCGAGGGGAC ACTGGAGGTG 360CTAGCAGTGA GGACAGCCTG ATGACCAGCT TCCTGCCAGG CCCTAAGCCT GGAGCTCCCT 420TCCCTAATGG ACACGTGGGT GCTGGAGGCA GTGGCCTGCT CCCACCTCCA CCCGCGCTCT 480GCGGGGCCTC TGCCTGTGAT GTCTCCGTAC GTGTGGTGGT GGGTGAGCCC ACCGAGGCCA 540GGGTGGTTCC GGGCCGGGGC ATCTGCCTGG ACCTCGCCAT CCTGGATAGT GCCTTCCTGC 600TGTCCCAGGT GGCCCCATCC CTGTTTATGG GCTCCATTGT CCAGCTCAGC CAGTCTGTCA 660CTGCCTATAT GGTGTCTGCC GCAGGCCTGG GTCTGGTCGC CATTTACTTT GCTACACAGG 720TAGTATTTGA CAAGAGCGAC TTGGCCAAAT ACTCAGCGTA GAAAACTTCC AGCACATTGG 780GGTGGAGGGC CTGCCTCACT GGGTCCCAGC TCCCCGCTCC TGTTAGCCCC ATGGGGCTGC 840CGGGCTGGCC GCCAGTTTCT GTTGCTGCCA AAGTAATGTG GCTCTCTGCT GCCACCCTGT 900GCTGCTGAGG TGCGTAGCTG CACAGCTGGG GGCTGGGGCG TCCCTCTCCT CTCTCCCCAG 960TCTCTAGGGC TGCCTGACTG GAGGCCTTCC AAGGGGGTTT CAGTCTGGAC TTATACAGG 1020AGGCCAGAAG GGCTCCATGC ACTGGAATGC GGGACTCTGC AGGTGGATTA CCCAGGCTC 1080GGGTTAACAG CTAGCCTCCT AGTTGAGACA CACCTAGAGA AGGGTTTTTG GGAGCTGAA 1140AAACTCAGTC ACCTGGTTTC CCATCTCTAA GCCCCTTAAC CTGCAGCTTC GTTTAATGT 1200GCTCTTGCAT GGGAGTTTCT AGGATGAAAC ACTCCTCCAT GGGATTTGAA CATATGAAA 1260TTATTTGTAG GGGAAGAGTC CTGAGGGGCA ACACACAAGA ACCAGGTCCC CTCAGCCCA 1320AGCACTGTCT TTTTGCTGAT CCACCCCCCT CTTACCTTTT ATCAGGATGT GGCCTGTTG 1380TCCTTCTGTT GCCATCACAG AGACACAGGC ATTTAAATAT TTAACTTATT TATTTAACA 1440AGTAGAAGGG AATCCATTGC TAGCTTTTCT GTGTTGGTGT CTAATATTTG GGTAGGGTG 1500GGGATCCCCA ACAATCAGGT CCCCTGAGAT AGCTGGTCAT TGGGCTGATC ATTGCCAGA 1560TCTTCTTCTC CTGGGGTCTG GCCCCCCAAA ATGCCTAACC CAGGACCTTG GAAATTCTA 1620TCATCCCAAA TGATAATTCC AAATGCTGTT ACCCAAGGTT AGGGTGTTGA AGGAAGGTA 1680AGGGTGGGGC TTCAGGTCTC AACGGCTTCC CTAACCACCC CTCTTCTCTT GGCCCAGCC 1740GGTTCCCCCC ACTTCCACTC CCCTCTACTC TCTCTAGGAC TGGGCTGATG AAGGCACTG 1800CCAAAATTTC CCCTACCCCC AACTTTCCCC TACCCCCAAC TTTCCCCACC AGCTCCACA 1860CCCTGTTTGG AGCTACTGCA GGACCAGAAG CACAAAGTGC GGTTTCCCAA GCCTTTGTC 1920ATCTCAGCCC CCAGAGTATA TCTGTGCTTG GGGAATCTCA CACAGAAACT CAGGAGCAC 1980CCCTGCCTGA GCTAAGGGAG GTCTTATCTC TCAGGGGGGG TTTAAGTGCC GTTTGCAAT 2040ATGTCGTCTT ATTTTTTTAG CGGGGTGAAT ATTTTATACT GTAAGTGAGC AATCAGAGT 2100TAATGTTTAT GGTGACAAAA TTAAAGGCTT TCTTATATGT TTA 2143 2152 base pairsnucleic acid single linear not provided 16 GGGGCTGTAC CAGGGCGTGCCCAGAGCTGA GCCGGGCACC GAGGCCCGGA GACACTATGA 60 TGAAGGCGTT CGGATGGGCAGCCTGGGGCT GTTCCTGCAG TGCGCCATCT CCCTGGTCTT 120 CTCTCTGGTC ATGGACCGGCTGGTGCAGCG ATTCGGCACT CGAGCAGTCT ATTTGGCCAG 180 TGTGGCAGCT TTCCCTGTGGCTGCCGGTGC CACATGCCTG TCCCACAGTG TGGCCGTGGT 240 GACAGCTTCA GCCGCCCTCACCGGGTTCAC CTTCTCAGCC CTGCAGATCC TGCCCTACAC 300 ACTGGCCTCC CTCTACCACCGGGAGAAGCA GGTGTTCCTG CCCAAATACC GAGGGGACAC 360 TGGAGGTGCT AGCAGTGAGGACAGCCTGAT GACCAGCTTC CTGCCAGGCC CTAAGCCTGG 420 AGCTCCCTTC CCTAATGGACACGTGGGTGC TGGAGGCAGT GGCCTGCTCC CACCTCCACC 480 CGCGCTCTGC GGGGCCTCTGCCTGTGATGT CTCCGTACGT GTGGTGGTGG GTGAGCCCAC 540 CGAGGCCAGG GTGGTTCCGGGCCGGGGCAT CTGCCTGGAC CTCGCCATCC TGGATAGTGC 600 CTTCCTGCTG TCCCAGGTGGCCCCATCCCT GTTTATGGGC TCCATTGTCC AGCTCAGCCA 660 GTCTGTCACT GCCTATATGGTGTCTGCCGC AGGCCTGGGT CTGGTCGCCA TTTACTTTGC 720 TACACAGGTA GTATTTGACAAGAGCGACTT GGCCAAATAC TCAGCGTAGA AAACTTCCAG 780 CACATTGGGG TGGAGGGCCTGCCTCACTGG GTCCCAGCTC CCCGCTCCTG TTAGCCCCAT 840 GGGGCTGCCG GGCTGGCCGCCAGTTTCTGT TGCTGCCAAA GTAATGTGGC TCTCTGCTGC 900 CACCCTGTGC TGCTGAGGTGCGTAGCTGCA CAGCTGGGGG CTGGGGCGTC CCTCTCCTCT 960 CTCCCCAGTC TCTAGGGCTGCCTGACTGGA GGCCTTCCAA GGGGGTTTCA GTCTGGACT 1020 ATACAGGGAG GCCAGAAGGGCTCCATGCAC TGGAATGCGG GGACTCTGCA GGTGGATTA 1080 CCAGGCTCAG GGTTAACAGCTAGCCTCCTA GTTGAGACAC ACCTAGAGAA GGGTTTTTG 1140 GAGCTGAATA AACTCAGTCACCTGGTTTCC CATCTCTAAG CCCCTTAACC TGCAGCTTC 1200 TTTAATGTAG CTCTTGCATGGGAGTTTCTA GGATGAAACA CTCCTCCATG GGATTTGAA 1260 ATATGAAAGT TATTTGTAGGGGAAGAGTCC TGAGGGGCAA CACACAAGAA CCAGGTCCC 1320 TCAGCCCACA GCACTGTCTTTTTGCTGATC CACCCCCCTC TTACCTTTTA TCAGGATGT 1380 GCCTGTTGGT CCTTCTGTTGCCATCACAGA GACACAGGCA TTTAAATATT TAACTTATT 1440 ATTTAACAAA GTAGAAGGGAATCCATTGCT AGCTTTTCTG TGTTGGTGTC TAATATTTG 1500 GTAGGGTGGG GGATCCCCAACAATCAGGTC CCCTGAGATA GCTGGTCATT GGGCTGATC 1560 TTGCCAGAAT CTTCTTCTCCTGGGGTCTGG CCCCCCAAAA TGCCTAACCC AGGACCTTG 1620 AAATTCTACT CATCCCAAATGATAATTCCA AATGCTGTTA CCCAAGGTTA GGGTGTTGA 1680 GGAAGGTAGA GGGTGGGGCTTCAGGTCTCA ACGGCTTCCC TAACCACCCC TCTTCTCTT 1740 GCCCAGCCTG GTTCCCCCCACTTCCACTCC CCTCTACTCT CTCTAGGACT GGGCTGATG 1800 AGGCACTGCC CAAAATTTCCCCTACCCCCA ACTTTCCCCT ACCCCCAACT TTCCCCACC 1860 GCTCCACAAC CCTGTTTGGAGCTACTGCAG GACCAGAAGC ACAAAGTGCG GTTTCCCAA 1920 CCTTTGTCCA TCTCAGCCCCCAGAGTATAT CTGTGCTTGG GGAATCTCAC ACAGAAACT 1980 AGGAGCACCC CCTGCCTGAGCTAAGGGAGG TCTTATCTCT CAGGGGGGGT TTAAGTGCC 2040 TTTGCAATAA TGTCGTCTTATTTATTTAGC GGGGTGAATA TTTTATACTG TAAGTGAGC 2100 ATCAGAGTAT AATGTTTATGGTGACAAAAT TAAAGGCTTT CTTATATGTT TA 2152 68 base pairs nucleic acidsingle linear not provided 17 AGCTCGGAAT TCCGAGCTTG GATCCTCTAGAGCGGCCGCC GACTAGTGAG CTCGTCGACC 60 CGGGAATT 68 68 base pairs nucleicacid single linear not provided 18 AATTAATTCC CGGGTCGACG AGCTCACTAGTCGGCGGCCG CTCTAGAGGA TCCAAGCTCG 60 GAATTCCG 68 24 base pairs nucleicacid single linear not provided 19 AGCGGATAAC AATTTCACAC AGGA 24 18 basepairs nucleic acid single linear not provided 20 TGTAAAACGA CGGCCAGT 1818 base pairs nucleic acid single linear not provided 21 TGTTCCTGCCCAAATACC 18 18 base pairs nucleic acid single linear not provided 22GGTCTGGTCG CCATTTAC 18 18 base pairs nucleic acid single linear notprovided 23 GGGGCAACAC ACAAGAAC 18 19 base pairs nucleic acid singlelinear not provided 24 TCAGCCCCCA GAGTATATC 19 18 base pairs nucleicacid single linear not provided 25 GCTCCATGCA CTGGAATG 18 18 base pairsnucleic acid single linear not provided 26 ACCCAGGACC TTGGAAAT 18 20base pairs nucleic acid single linear not provided 27 ACACCCTAACCTTGGGTAAC 20 20 base pairs nucleic acid single linear not provided 28CCTAGAAACT CCCATGCAAG 20 18 base pairs nucleic acid single linear notprovided 29 TGGCAGCAAC AGAAACTG 18 18 base pairs nucleic acid singlelinear not provided 30 ACTATCCAGG ATGGCGAG 18 18 base pairs nucleic acidsingle linear not provided 31 TGATTGCTCA CTTACAGT 18 18 base pairsnucleic acid single linear not provided 32 TGGTTAGGGA AGCCGTTG 18 18base pairs nucleic acid single linear not provided 33 AGCCCAATGACCAGCTAT 18 22 base pairs nucleic acid single linear not provided 34TTCCAAATGC TGTTACCCAA GG 22 24 base pairs nucleic acid single linear notprovided 35 GGTGCTCCTG AGTTTCTGTG TGAG 24 255 amino acids amino acidsingle linear None not provided 36 Gly Leu Tyr Gln Gly Val Pro Arg AlaGlu Pro Gly Thr Glu Ala Arg 1 5 10 15 Arg His Tyr Asp Glu Gly Val ArgMet Gly Ser Leu Gly Leu Phe Leu 20 25 30 Gln Cys Ala Ile Ser Leu Val PheSer Leu Val Met Asp Arg Leu Val 35 40 45 Gln Arg Phe Gly Thr Arg Ala ValTyr Leu Ala Ser Val Ala Ala Phe 50 55 60 Pro Val Ala Ala Gly Ala Thr CysLeu Ser His Ser Val Ala Val Val 65 70 75 80 Thr Ala Ser Ala Ala Leu ThrGly Phe Thr Phe Ser Ala Leu Gln Ile 85 90 95 Leu Pro Tyr Thr Leu Ala SerLeu Tyr His Arg Glu Lys Gln Val Phe 100 105 110 Leu Pro Lys Tyr Arg GlyAsp Thr Gly Gly Ala Ser Ser Glu Asp Ser 115 120 125 Leu Met Thr Ser PheLeu Pro Gly Pro Lys Pro Gly Ala Pro Phe Pro 130 135 140 Asn Gly His ValGly Ala Gly Gly Ser Gly Leu Leu Pro Pro Pro Pro 145 150 155 160 Ala LeuCys Gly Ala Ser Ala Cys Asp Val Ser Val Arg Val Val Val 165 170 175 GlyGlu Pro Thr Glu Ala Arg Val Val Pro Gly Arg Gly Ile Cys Leu 180 185 190Asp Leu Ala Ile Leu Asp Ser Ala Phe Leu Leu Ser Gln Val Ala Pro 195 200205 Ser Leu Phe Met Gly Ser Ile Val Gln Leu Ser Gln Ser Val Thr Ala 210215 220 Tyr Met Val Ser Ala Ala Gly Leu Gly Leu Val Ala Ile Tyr Phe Ala225 230 235 240 Thr Gln Val Val Phe Asp Lys Ser Asp Leu Ala Lys Tyr SerAla 245 250 255 44 amino acids amino acid single linear None notprovided 37 Tyr His Arg Glu Lys Gln Val Phe Leu Pro Lys Tyr Arg Gly AspThr 1 5 10 15 Gly Gly Ala Ser Ser Glu Asp Ser Leu Met Thr Ser Phe LeuPro Gly 20 25 30 Pro Lys Pro Gly Ala Pro Phe Pro Asn Gly His Val 35 4027 amino acids amino acid single linear None not provided 38 Arg Val ValVal Gly Glu Pro Thr Glu Ala Arg Val Val Pro Gly Arg 1 5 10 15 Gly IleCys Leu Asp Leu Ala Ile Leu Asp Ser 20 25 27 amino acids amino acidsingle linear None not provided 39 Gly Leu Tyr Gln Gly Val Pro Arg AlaGlu Pro Gly Thr Glu Ala Arg 1 5 10 15 Arg His Tyr Asp Glu Gly Val ArgMet Gly Ser 20 25 8 amino acids amino acid single linear None notprovided 40 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 21 amino acids aminoacid single linear None not provided 41 Glu Gln Lys Leu Ile Ser Glu GluAsp Leu Asn Met His Thr Glu His 1 5 10 15 His His His His His 20

We claim:
 1. A polypeptide with an amino acid sequence selected from thegroup consisting of SEQUENCE ID NO 36; SEQUENCE ID NO 37; SEQUENCE ID NO38; SEQUENCE ID NO
 39. 2. The polypeptide of claim 1, wherein saidpolypeptide is produced by recombinant techniques.
 3. The polypeptide ofclaim 1, wherein said polypeptide is produced by synthetic techniques.4. An antibody which specifically binds to at least one epitope, whereinsaid epitope has an amino acid sequence selected from the groupconsisting of SEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38,SEQUENCE ID NO
 39. 5. An assay kit for determining the presence of anantigen or antibody in a test sample, said assay kit comprising acontainer containing a polypeptide having an amino acid sequenceselected from the group consisting of SEQUENCE ID NO 36, SEQUENCE ID NO37, SEQUENCE ID NO 38, SEQUENCE ID NO
 39. 6. The assay kit of claim 5,wherein said polypeptide is attached to a solid phase.
 7. The assay kitof claim 5 further comprising a container with tools useful forcollection of said sample, wherein the tools are selected from the groupconsisting of lancets, absorbent paper, cloth, swabs and cups.
 8. Thetest kit of claim 5 further comprising a container with tools useful forcollection of said sample, wherein the tools are selected from the groupconsisting of lancets, absorbent paper, cloth, swabs and cups.